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Heavy metal accumulation by transformed and non transformed AM fungi

Three groups of Gi. rosea spores were provided by the consortium: non-bombarded (NB), bombarded with an empty vector (BP) and bombarded with the GmarMT1 gene (BG). The spores were placed on sterile filter disks on sterile sand moistened with different metal solutions: Cd (4,5, 45 and 450 µM), Zn (75, 750 and 7500 µM) and Cu (50, 500 and 5000 µM) and checked for germination. After 4 weeks approximately 30 spores were stained with INT to check their viability, divided into live (positive reaction) and dead (negative reaction) and counted.

Generally, in the control group, the bombarded spores (BP and BG) showed decreased viability compared to non-bombarded spores, although this difference was not significant. The same tendency was noted in the Cd-treated groups (4.5 and 450 µM). In the case of Zn, the tendency was the opposite: BG spores showed the highest viability. In the Cu-treated group, the situation was similar at the low and medium Cu concentrations (the BG group spores). Spores at the lowest Cu and Zn concentrations died due to parasitic attack. At the highest Cu concentration the overall viability was very low and the spores turned dark.

It was also observed that the dark spores incubated with high Cu concentrations (5000 µM) showed a thickening of the cell wall. Dithizone and rhodizonate treatment of unstained spores (highest concentrations) showed a positive reaction in the case of Cd and Zn-treated groups and a much weaker reaction in the Cu-treated group. These dyes are excellent indicators of the accumulation of heavy metals in tissues, especially when used for pilot screening of the material. Spores that formed at least 500 öm long hyphae were removed from the filters and attached to carbon holders. The mycelium and the attached spores, cut open to facilitate drying up, were air-dried in order to prevent element loss due to lyophilisation, coated with carbon and analysed with EDS coupled to SEM.

No statistically important differences in Cd content in the mycelium and both spore layers were found in spores germinated in 4,5 µM Cd solution. The differences became clear only at the higher Cd levels. At the highest Cd concentration (450µM) the highest content of Cd was found within the mycelium. The high Cd level was accompanied by a high content of S. S can come from the sulphate salts of the metals used in the experiment. While the first two batches were analysed, in the 450µM Cd solution, only spores bombarded with the MT gene germinated. This suggested the successful improvement of resistance to Cd. The same tendency was noted for Cu. The high variation between individual spores can be explained by the effectiveness of transformation of about 50%. No differences were noted in the case of Zn.

The third batch of spores appeared to be of the high vitality and the germination % was very high. Some of spores bombarded with the GmarMT1 gene clearly contained higher levels of Cd, although others accumulated much less that again could be caused either by the fact that not all spores were modified or by the fact that some hyphae were growing above the surface of the filter and had little contact with Cd. The clear increase of the mycelium growth rate was found in the case of the GmarMT1 gene bombarded spores that were exposed to lower Cd solutions within microtiter plates.

More information on the Genomyca -project can be found at: http://www.dijon.inra.fr/genomyca/

Reported by

Jagiellonian University
Institute of Botany, Ul. Lubicz 46
31-512 Krakow
Poland
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