Drought stress response of tetraploid hybrid aspen (Populus tremula L. x P. tremuloides Michx.) of protoplast fusion experiments)

Poplar (Populus) varieties that are planted in short rotation coppice are supposed to show high biomass production, the ability to propagate via stem cuttings but also drought tolerance because drought is a very important abiotic stressor that can negatively influence the plants' growth, vitality and productivity. For polyploid varieties of various species as crop species (Triticum), herbaceous species (Lonicera, Spathiphyllum, Nicotiana) as well as in tree species (Betula) higher tolerance towards stresses as, for example, drought was reported. Hybrid aspen Populus tremula (L.) x P. tremuloides (Michx.) that belong to the section Populus exhibit economically viable increase on soils where other tree species fail but miss the propagation via stem cuttings. Poplar species of the sections Tacamahaca and Aigeiros as, for example, P. nigra (L.) and P. trichocarpa (Torr. & Gray) x P. deltoides (Bartram ex Marsh) can be propagated via stem cuttings. To overcome crossing incompatibilties somatic hybridization was applied to P. tremula x P. tremuloides ('Munden 2') as one fusion partner and P. x canescens (INRA clone No. 717 1‑B4), P. nigra or P. trichocarpa x P. deltoides (B19) as the second fusion partner. According to the leaf morphology and microsatellite analysis the obtained fusion lines were assigned to the original clone P. tremula x P. tremuloides ('Munden 2') and showed a tetraploid set of chromosomes. In this dissertation, the main aim was to investigate the drought responses of the tetraploid hybrid aspen lines in comparison to the diploid original line P. tremula x P. tremuloides ('Munden 2'). Therefore, the following questions were addressed. (i) Do the tetraploid hybrid aspen lines perform better under drought than the diploid original line regarding e.g. the water consumption and the leaf vitality? (ii) Do the tetraploid hybrid aspen lines distinguish from the diploid original line in morphological traits as stomatal morphology, height, stem biomass and leaf area? (iii) Is the better drought performance in the tetraploid hybrid aspen lines due to the increased ploidy level, i.e. do the tetraploid hybrid aspen lines show better drought performance as higher survival rates, a delay in leaf wilting and higher relative leaf water content than the diploid “fusion” lines? (iv) Do the tetraploid hybrid aspen show early physiological changes in the relative leaf water content, the carbohydrate concentration and the stomatal conductance? (v) Are there any genetic modifications apart from the tetraploidy like duplications or deletions that might influence the drought performance? In a first experiment, four hybrid aspen lines were obtained. Two of them showed lower water consumption relative to height and three delayed leaf wilting compared to the diploid original line. Furthermore, the tetraploid hybrid aspen lines exhibited lower stomatal density, increased stomatal length, comparable or lower height, stem biomass and total leaf area and enhanced leaf mass per area when compared to the diploid original line. In a second experiment, four diploid “fusion” lines and 16 tetraploid fusion lines were screened for their drought performance. The tetraploid fusion lines showed enhanced survival rates over the diploid lines but exhibited decreased height. With regard to height 11 tetraploid fusion lines exhibited delayed total leaf wilting while none of the four diploid ”fusion” lines showed a delay in total leaf wilting with regard to the height when compared to the diploid original line. In a third experiment, two lines were selected from the second experiment that showed a delay in total leaf wilting with regard to height but comparable height growth to the diploid original line. Here, physiological traits like the water consumption and physiological responses like the relative leaf water content, the stomatal conductance and the carbohydrate content were investigated in a time course. A lower water consumption was observed in both tetraploid fusion lines in comparison to the diploid original line resulting in lower drought stress of the tetraploid fusion lines. The finding of lower water consumption in the tetraploid fusion lines was consistent with the increased soil moisture levels compared to the diploid original line. On the other hand, a lower water consumption would be in accordance with a lower stomatal conductance that was not observed in general for the tetraploid fusion lines. The tetraploid fusion lines were able to maintain the stomatal conductance relative to the control at relative soil moisture levels where the diploid original line had already closed their stomata. This finding supports a higher drought tolerance for the tetraploid fusion lines. Genetic modifications such as duplications and deletions were detected using copy number variation analysis. Whole genome sequencing was applied to three tetraploid fusion lines and the diploid original line. The three tetraploid fusion lines exhibited growth comparable to that of the diploid original line but also delayed total leaf wilting with regard to height. The copy number variation analysis revealed only three segments that were altered in all tetraploid fusion lines compared to the diploid line. Genes that were putatively encoded by these segments were not annotated. One tetraploid fusion line showed a high number of 90 segments that were increased in this line but neither in the diploid original nor in the other two sequenced tetraploid fusion lines. Although this line showed better drought performance than the diploid original line the water consumption, the leaf vitality and the stomatal conductance did not differ from the other tested tetraploid fusion line (experiment 3). This finding indicates that the drought response is rather due to the tetraploidy than to other genetic modifications. Nevertheless, a functional characterization of the three segments that were altered in all tetraploid fusion lines in comparison to the diploid line is useful and should be conducted in furture studies as drought-relevant genes might be affected. The relevance to drought of these putative genes could be tested by exposing knock-out mutants to dry conditions. Finally, it should be noted that the heterofusion frequency was very low with a few hybrids dying already in the in vitro stage. The fusion products originated from a joint project with several hundred fusion lines that were genetically characterized and only a few revealed as heterofusions. The heterofusion frequencies in other species (Cyclamen, Brassicaceae, Fabaceae, Poaceae, Solanaceae) is low as well. Since in this dissertation homofusion lines were investigated only, the propagation via stem cuttings was not investigated but I focused on drought responses of the tetraploid hybrid aspen lines. In conclusion, morphological changes and higher drought tolerance were observed in the hybrid aspen lines with increased ploidy level. This renders them to be planted on sites where water is a limiting factor and high failure of plants due to drought is expected.