Browsing by Subject "Tissue culture"

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Study of the natural resistance towards apple proliferation disease and establishment of an in vitro resistance screening system in view of the development of resistant apple rootstocks
(2007) Bisognin, Claudia; Reustle, Götz
Apple proliferation (AP) is an economically important disease of apple which occurs in all countries of central and southern Europe. All currently grown cultivars and rootstocks are susceptible to the disease and no curative treatments are applicable. AP is caused by a phytoplasma, Candidatus Phytoplasma mali, which is restricted to the phloematic tissue of the plant. Ca. P. mali is naturally spread by psyllid vectors and by root bridges as well as by man through infected planting material. An efficient control of the disease is hampered by these different ways of transmission. The objective of the thesis was therefore to evaluate a strategy for a long-term solution to AP based on natural resistance. This resistance has been detected in the wild apomictic species Malus sieboldii and in first and second generation hybrids of M. sieboldii. Although the obtained progeny turned out to be too vigorous for modern apple culture, a certain number of genotypes remained resistant to AP. While phytoplasmas colonise constantly the roots of infected trees, infections in the susceptible cultivar are eliminated each year during the renewal of the phloem in early spring. A resistance strategy towards AP can therefore be solely based on AP-resistant rootstocks in order to prevent the re-colonisation of the canopy in spring. The first part of the thesis was concentrated on the re-evaluation of the AP resistance in M. sieboldii and its hybrids in a 12-years field trial under natural infection pressure at BBA Dossenheim. The annual data for symptom recording and fruit size were analysed as cumulative disease index and cumulative undersized fruit index, respectively. By the end of the trial the phytoplasma concentration in roots and shoots was analysed by quantitative real-time PCR. The results confirmed previous data that M. sieboldii and its hybrids exhibit resistance towards AP. Infected trees of these genotypes had low concentrations of phytoplasmas in the roots and showed almost no symptoms and no undersized fruits. Contrary, previously as resistant classified genotypes derived from M. sargentii reacted highly susceptible. As individual trees of the resistant genotypes showed an altered behaviour, molecular analyses were performed to verify if the seed propagated, apomictic material used for the trial was really true-to-type. For this analysis, co-dominant microsatellite (SSR) markers were used which were derived from published work. However, for each M. sieboldii genotype suitable, polymorphic markers had to be selected. This analysis revealed that apomixis was not complete and that a varying percentage of progeny of the different genotypes was recombinant due to open pollination. As the resistant M. sieboldii genotypes were too vigorous for modern apple culture, new breedings were carried out with these genotypes in combination with dwarfing rootstock genotypes such as M9. More than 3.000 seedlings have been produced in 17 cross combinations. All seedlings were examined by microsatellite analysis in order to distinguish recombinant from non-recombinant, apomictic progeny. SSR markers were also useful in determining the ploidy level of the parents and their progenies. These results were confirmed by flow cytometric analysis. The recombinant progeny is currently being evaluated in the field for its AP resistance. As the screening of resistance towards AP in the field necessitates several years of observation, an alternative in vitro method was developed. This method is based on in vitro graft-inoculation of the genotype to test. As a prerequisite in vitro cultures of all parental genotypes of the breeding program were established. For each genotype the optimal culture medium was defined. Thus, efficient micropropagation and in vitro rooting protocols were established for the M. sieboldii genotypes which are difficult to root under normal nursery conditions. The established protocols enable a large scale production of these genotypes on a commercial scale. The in vitro resistance screening method allows the evaluation of a given genotype under standardized conditions by using repetitions of micro-grafts. The quality of the grafts, the mortality, the transmission rates of the phytoplasmas as well as the concentration of the phytoplasmas in the inoculated genotypes was determined. The results obtained by qPCR showed that the phytoplasma concentration in resistant genotypes was significantly lower than in susceptible ones. Whereas susceptible genotypes exhibited stunted growth and proliferation symptoms in vitro the resistant genotypes had a phenotype almost comparable to the healthy control. Interestingly, significant differences in phytoplasma concentration could be found between two different Ca. P. mali subtypes used in the experiments. The results demonstrated that the method enables a reliable result 3 months p.i. and can be used to evaluate the virulence of different Ca. P. mali strains.
Untersuchungen des Einflusses von Chemotherapie auf Tumor-assoziierte Fibroblasten bei Karzinomen der Lunge und Brust in vivo und in verschiedenen ex vivo Modellen
(2009) Sonnenberg, Maike; Blum, Martin
Carcinomas are complex tissues in which the genetically altered epithelial tumor cells interact with their microenvironment, which plays a pivotal role in growth and survival of the whole tumor. Therefore, also the response of the tumor stroma may be important for the therapeutic success. The aim of the study was to clarify how tumor-associated fibroblasts (TAFs) respond to chemotherapy. We established a grading system for the activity of the TAFs based on cell counts and cell morphology within neoadjuvant treated breast cancer specimens. We compared both the stromal compartment and the tumor compartment before and after therapy to evaluate the response to chemotherapy. In this in vivo study a response of the stromal compartment was detected in samples with a high stromal activity grade before chemotherapy. To investigate the direct, more acute response of TAFs we established different ex vivo systems. On the one hand, the response to chemotherapy was tested in primary isolated TAFs from tumor tissue. On the other hand, a tissue slice culture was established to investigate the direct effect of the chemotherapy on TAFs and tumor cells within their intact microenvironment. For the cell culture experiments freshly isolated TAFs from breast and lung tumors were used. The chemosensitivity to Cisplatinum and Paclitaxel of the primary isolated TAFs was determined and compared with a panel of established human tumor cell lines. The TAFs turned out to be resistant to Paclitaxel, whereas they showed a heterogeneous response to Cisplatinum. One reason for this heterogeneous response to Cisplatinum could be the existence of somatic mutations and/or polymorphisms within DNA-damage response genes. Somatic mutations within the p53 tumor suppressor gene play a critical role in the response of tumor cells to chemotherapeutics. In this study, however, no somatic mutation was detected in any of the TAFs from lung. In other studies, different polymorphisms could be correlated with the sensitivity to cytotoxic stress. In this study, a non-significant trend towards the chemosensitivity of TAFs from lung carcinomas and the p53-Arg72Pro polymorphism could be observed, whereas the ERCC1-Asn118Asn und Mdm2-T/G309 polymorphisms had no influence on the chemosensitivity. The chemosensitivity of the TAFs was also determined within their microenvironment on the basis of tissue culture experiments performed with breast and lung carcinomas. Tumor tissue slices from breast carcinomas were treated for 72 h with Paclitaxel, whereas the tissue slices from lung carcinomas were treated with Cisplatinum. To investigate the response of the different cell types to the tested chemotherapeutic drugs within the intact microenvironment, the tissue slices were fixed and stained immunhistochemically for proliferative active and dead cells. Half of the cultivated tissue slices from breast carcinomas showed a decrease in cell counts of proliferating cells, whereas half of the cases showed an increase of dead cells after the treatment with Paclitaxel in tumor and stroma cells. The tissue slices of the lung carcinomas showed a decrease in proliferating tumor cells after Cisplatinum treatment in comparison to the untreated controls. The stroma compartment showed no proliferative activity so one could not expect a response regarding a decrease of proliferating stroma cells. Two cases showed a response to Cisplatinum regarding an increase in cell death in both cell compartments. Short term exposure to Paclitaxel led to a parallel reaction of both tumor and stromal cells in tissue culture experiments, whereas isolated TAFs from breast tumors turned out to be resistant to Paclitaxel. In contrast, isolated TAFs from lung carcinomas are significantly more sensitive to Cisplatinum than TAFs within their intact microenvironment. Consequently, the microenvironment of the tumor plays a crucial role in chemosensitivity. The central role of the microenvironment for response of TAFs to cytotoxic drugs is also demonstrated by the results obtained with lung cancer tissues. These observations indicate that, in addition to intrinsic factors, the microenvironment determines the sensitivity of TAFs to cytotoxic therapy. In summary, this work demonstrates for the first time, not only the tumor cells but also the stromal cells are an important target for the chemotherapy. Intrinsic and extrinsic factors play an important role in chemosensitivity. On the one hand, the tumor microenvironment is responsible for therapy response of TAFs and on the other hand the genotype may play a crucial role in chemosensitivity. Both, tumor and stroma cells react in parallel to chemotherapy, so you can assume that both cell types are responsible for the chemosensitivity. Some therapeutic approaches could be the inhibition of either the tumor or stromal cell compartment to increase the chemosensitivity of the whole tumor.