Maria Katarzyna Wojciechowicz

Cytological analysis of hybrid embryos of intergeneric crosses between Salix viminalis and Populus species.

Intergeneric hybridisation between Salix viminalis L. as the female and four Populus species (Populus trichocarpa, P. tremula, P. × canadensis and P. simonii) as male pollen donors was performed by in vitro stigma pollination. To overcome postzygotic barriers, transfer of hybrid embryos to new medium is necessary. We carried out detailed ultrastructural analyses to establish: (i) at which stage of embryo development the first signs of programmed cell death (PCD) could be detected; and (ii) at which stage the lack of serious or irreversible changes guaranteed that advanced development of hybrid plants could occur after embryo rescue. Transmission electron microscopy and confocal laser scanning microscopy revealed the presence of both developing and degenerating embryos. Developing globular, heart-shaped, and early cotyledonary embryos contained cells of correct ultrastructure. The only sign of intergeneric hybridisation was a delay in development for a few days, in comparison with control embryos. The earliest indicators of embryo degeneration were noted at 9 days after pollination (DAP). The most common indicators were excessive embryo vacuolisation, which was characterised by a large number of vesicles and formation of small vacuoles, as well as enlarged central vacuoles. Extended plastid thylakoids, folding of the cell wall, and autophagosomes were observed. Our detailed investigation of PCD in hybrid embryos enabled us to conclude that the embryo rescue technique was most effective in intergeneric willow × poplar crosses if applied between 9 and 16 DAP.

Effects of Daphnia exudates and sodium octyl sulphates on filament morphology and cell wall thickness of Aphanizomenon gracile (Nostocales), Cylindrospermopsis raciborskii (Nostocales) and Planktothrix agardhii (Oscillatoriales)

ABSTRACT Grazing is recognized as one of the selective factors shaping the morphology and physiology of cyanobacteria. A recent study has shown that the filamentous cyanobacterium Aphanizomenon gracile strain SAG 31.79 thickened in the presence of Daphnia (Cladocera) and its exudates. The aims of our study were: (1) to determine whether this type of response to Daphnia cues is common for other strains of A. gracile, and other species of filamentous cyanobacteria, (2) to test whether the response is due to nutrients recycled by Daphnia, or kairomone induced, and (3) whether it is related to toxin production. Prior to the experiment, cyanobacterial strains were inspected using chromatographic methods for the presence of two toxins, cylindrospermopsin (CYN) and three homologues of microcystin (MC-RR, MC-YR, MC-LR). HPLC analyses showed that all strains were free of cylindrospermopsin, whereas microcystins were detected only in one strain (Planktothrix agardhii). We then tested whether Daphnia exudates can cause thickening of cyanobacterial filaments, which would suggest the morphological changes in cyanobacterial filaments are caused by recycled nutrients. Cyanobacteria were also exposed to sodium octyl sulphate (a commercially available Daphnia kairomone). Transmission electron microscopy (TEM) was used to check whether Daphnia exudates and sodium octyl sulphate trigger thickening of cyanobacterial cell walls, which would be a defence mechanism against grazing. The TEM analysis revealed no significant effect of either Daphnia exudates or kairomone (sodium octyl sulphate) on the cell wall thickness of cyanobacteria. However, our study showed that Daphnia exudates triggered filament thickening in nostocalean cyanobacteria, while filaments of the oscillatorialean strain P. agardhii did not show this response. It was also demonstrated that sodium octyl sulphate alone can also cause filament thickening, which suggests that this might be a specific defence response to the presence of grazers.

Solitary terminal cells of Aphanizomenon gracile (Cyanobacteria, Nostocales) can divide and renew trichomes

An attempt was made to find evidence that morphologically distinct terminal cells of filamentous cyanobacterium Aphanizomenon gracile strain CCALA 8 are capable of dividing and forming trichomes. Based on our current knowledge, the division of morphologically diversified terminal cells is possible in nostocalean cyanobacteria. However, this process has been observed only in a few species. Terminal cells of A. gracile differ morphologically from other vegetative cells of a trichome, as they are not hyaline and can sometimes be found as solitary cells in cultures. Hence, it was reasonable for us to suspect that these cells are capable of dividing and forming trichomes. We observed terminal cells under a light and transmission electron microscope. Microscopic observations revealed that the septum formed in both solitary terminal cells and in terminal cells attached to trichomes. Our study is the first to demonstrate division and renewal of trichomes in terminal cells of A. gracile. Previously, such mode of reproduction was described only for another nostocalean cyanobacterium Raphidiopsis mediterranea. Moreover, our findings further emphasize the variability among members that belong to the genus Aphanizomenon , in which a type species (A. flos‐aquae) has hyaline cells incapable of dividing and renewing trichomes, while A. gracile can additionally propagate by solitary terminal cells division. This additional feature distinguishing A. gracile from typical species of Aphanizomenon, such as A. flos‐aquae, might be valuable for resolving taxonomic position of the species considering ambiguous genetic relationship between A. gracile and A. flos‐aquae.