Phytoextraction of heavy metal from contaminated soils using genetically modified plants

The use of plants to clean-up soils contaminated with trace elements could provide a cheap and sustainable technology for phytoremediation. The introduction of novel traits into plants that produce high levels of biomass using a transgenic approach is a promising strategy for the development of effective phytoremediation technologies (Krämer, et al. 2001). This thesis describes the use of recombinant Chinese hamster and Saccharomyces cerevisiae MTIIs for generating transgenic tobacco plants with improved extraction, accumulation and tolerance to heavy metals. The ChMTII and ScMTII were amplified from cDNA and subcloned into plant expression vectors for transient expression in the vacuole. Detailed characterization of recombinant vacuolar targeted ChMTII-GFP fusion; ChMTII and ScMTII were carried out on stably transformed tobacco plants obtained by Agrobacterium-mediated transformation. The MTIIs were cloned into the pTRA-kc plant expression vector and analyzed for their stability and accumulation levels in the plant vacuole. Lines with the highest accumulation levels were selected for establishing homozygous lines. Functional expression of ChMTII and ScMTII in the vacuole of transgenic tobacco plants was confirmed by immunoblot and immunogold analyses. Immunoblot analysis indicated that high accumulation of the recombinant proteins was achieved in tobacco plants transformed with the cV-ChMTII-GFP construct, whereas the lower protein accumulation was observed for the S-ScMTII. Immunogold labelling experiments were carried out on transiently and stably transformed tobacco plants demonstrated that MTIIs localized to the vacuole. The T2 generation of tobacco lines expressing recombinant cV-ChMTII-GFP (line 13/3) as well as the T1 generation of the S-ChMTII (line 18) and S-ScMTII (line 24) were used for evaluating in vivo protein function with two cultivation system: hydroponic culture and soil. The levels of accumulation for the recombinant MTIIs in shoots and roots decreased with increasing concentration of Cd supplied with the nutrient solution. Transgenic plants that were transformed with the cV-ChMTII-GFP construct showed the highest expression of MTII protein in shoots and roots and a higher degree of tolerance to Cd than plants transformed with the S vacuolar targeting cassettes or than wild type plants used as control. The cV-ChMTII-GFP transgenic plants which accumulated high levels of MTII proteins in shoots and roots showed high tolerance to Cd toxicity with high biomass production in hydroponic experiments. Cd tolerance level was up to 50µM Cd for transgenics and wild type tobacco plants. Above this Cd concentration, the production of plant biomass, the accumulation of Cd and the degree of tolerance to the metal were decreasing. To determine the exact concentration threshold for Cd tolerance in the transgenic plants as compared to wild type tobacco plants additional experiment should be carried out. Ideally, the 50-100µM Cd range should be tested in hydroponic experiments. The transgenic and wild type plants in soil experiments didn’t show any toxicity symptoms within a treatment range from 0 to 30 ppm Cd. The dry matter of wild type and transgenic tobacco plants was only slightly affected by the treatment with Cd. Plants transformed with the cV-ChMTII-GFP cassette showed the highest dw compared to the other transgenic lines. However, wild type and transgenic plants showed no significant differences in the dw of leaves, stems and flowers. Further experiments should be carried out with higher cadmium dosage to identify those transgenic lines with improved ability to accumulate and tolerate heavy metals. The data presented in this study demonstrated that heterologous expression and subcellular targeting of MTIIs in tobacco plants was successfully achieved. The results of the hydroponics and soil experiment showed that the characterization of the transgenic tobacco plants might require further studies. However these data indicate that compared to traditional extraction approaches; phytoremediation represents a promising and environmental friendly tool to clean heavy metal from contaminated soils at reduced costs