Partner und Internationale Organisationen
(Englisch)
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A,B,CH,CZ,D,DK,E,F, FIN GR, H, I, IRL,N, NL P,PL,S, SI,SK, UK
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Abstract
(Englisch)
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Hypericum perforatum is a traditional medicinal plant, which received great attention in recent years by the pharmaceutical industry because of its antidepressive action. As a consequence high amounts of plant raw material is needed. However, suitable Hypericum varieties are the prerequisite for a successful cultivation. Hypericum perforatum is a tetraploid perennial herb native to Europe. The predominate type of reproduction is apomixis. The aim of the project is the opimization of the in vitro androgenesis of Hypericum perforatum in order to produce di-haploid and mono-haploid regenerants. The androgenic origin of the regenerants will be verified by molecular markers. These plants will be used to study the relationship between ploidy level and mass production, secondary metabolite production and fertility in order to evaluate their usefulness in practical breeding programs. For the development of di-haploid plants we first optimised the in vitro culture of entire anthers. Anthers (uninucleated and binucleated microspores) of ten different Hypericum genotypes were cultivated on MS medium differing in the combination of cytokinins (kinetin, BAP) and auxins (2,4-D, NAA) for callus induction. After 3 weeks first calli were observed and put on regeneration medium containing different levels (1mg/l or 2.5mg/l) of cytokinins (kinetin or BAP). After shoot induction, calli as well as isolated shoots were transferred to half concentrated hormone free MS medium with 10g/l sucrose for root induction and then transferred to soil. Different genotypes varied very much in their response to anther culture. Depending on the genotype we obtained up to 90 calli/100 anthers on MS medium with 0.5 mg/l BAP and 0.5 mg/l NAA. Regeneration medium containing 2.5 mg/l BAP yielded up to 84 shoots/100 calli resulting in a maximum of 75 shoots/100 cultivated anther. 71% of the shoots placed on hormone free medium formed roots. 312 regenerants were transferred to soil. 63% of these regenerants survived and developed to normal plants with fertile flowers. Before the regenerants were put in soil, leaf material of 54 plants were harvested for DNA extraction. Genetic analysis of the these regenerants were perforemd by RAPD analysis. Comparison of the regenerats with the donor plants revealed that 50 out of 54 regenerants were genetically identical to the donor plant. Thus, the majority of the regenerants were presumably derived from somatic anther tissue. Therefore we concentrated further activities on isolated microspore regeneration. After a cold pretreatment (4°C for 7 days) anthers were isolated from 15-20 flower buds. Microspores were collected by cutting the anthers in wash medium, removing the anther debris by filteration and concentrating the microspores by centrifugation. Survival rate of the microspores varied between 15 to 60%. Isolated microspores were cultured either on solid or liquid MS medium using different phytohormones and different concentrations of osmoltyicum (sucrose, polyethylenglycol, maltose). Best results were obtained using liquid medium. Although most microspores died during the culture, some started to grow and first cell divisions were observed after 14-20 days of culture. However, after a few cell divisions they stopped their growth. In the succeeding experiments we tested the isolated microspores of eight donor plants on 21 different liquid medium based on protocols of Murashige and Skoog (1962; MS), Chupeau (1993, BS), Gambor et al. (1968, B5) and Franklin et al. (1991, SI) with different concentrations of sucrose, maltose and mannitol with a final osmolarity ranging from 100 to 1050 mosm/l. The concentration of phytohormones 0.5 mg/l 2,4-D, 1.5mg/l NAA and 1.0 mg/l BA was identical in each medium. Isolation and inoculation of the microspores were done as described above. The survival rate of the microspores immediately after isolation ranged from 31 to 66% depending on the genotype of the donor plant. Based on the integrity of the microspores after 7 to 14 days of culture we selected four different media: BS (590 mosm/l), B5 (674 mosm/l) and SI (350 mosm/l) each with 20g/l sucrose and 80g/l mannitol (600 mosm/l) and SI with 10g/l sucrose and 40g/l mannitol (350 mosm/l). On these four media we tested the influence of a cold treatment before isolation, the microspore density during inoculation, the co-culture of pistil together with microspores and the influence of eight donor plants on microspore regeneration. First cell divisions were observed after 3-5 weeks and first microcalli after 6-7 weeks. Microcalli were obtained on each medium from microspores of only four donor plants. The percentage of petri dishes with microcalli (PDM) ranged from 10% (donor 2) to 78% (donor 1). The highest percentage of PDM was observed on the B5 medium (45%) compared to 10% on SI medium and at a concentration of 20'000 microspores per ml. Seven days of cold treatment before isolation of the microspores doubled the occurence of microcalli (32% vs. 14%). Best results were obtained with the co-culture of isolated microspores with the pistil of hypericum. This improved the percentage of PDM from 7% up to 40% independent of medium and cold treatment. For the best combination : donor plant 1, medium B5, cold treatment and pistil co-culture we obtained 4 microcalli per 20'000 isolated microspores. The microcalli were transferred recently on solid medium and continue to grow. Shot and root regeneration are expected the next months.
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