Lead phytoextraction by scented Pelargonium cultivars:udSoil-plant interactions and tool development for understanding lead hyperaccumulation

摘要

Metal removal from contaminated soils using plants can provide an environment friendly solution. However, its successful application on a large scale is still limited due to unavailability of plants with desired set of characteristics i.e. hyperaccumulation, high biomass and rapid growth. The objective of this work was to assess the potential of scented Pelargonium cultivars for lead (Pb) extraction under field conditions, plant induced rhizosphere changes, soil factors influencing availability of Pb and to develop an efficient genetic transformation protocol for the selected cultivars. Of the six scented Pelargonium cultivars field-tested, three cultivars (Attar of Roses, Clorinda and Atomic Snowflake) accumulated more than 1000 mg Pb kg-1 DW, with high biomass reaching up to 45 tons ha-1 y-1 dry matter. During assays in controlled conditions, Attar of roses (Pb hyperaccumulator) significantly acidified its rhizosphere and increased Dissolved Organic Carbon (DOC) concentration as compared to Concolor Lace (non-accumulator), probably due to enhanced exudation in response to the metal stress. Lead concentrations in both cultivars were best correlated with CaCl2 extracted Pb. Extended X-ray Absorption Fine Structure (EXAFS) and Environmental Scanning Electron Microscopy-Energy Dispersive x-ray Spectroscopy (ESEM-EDS) demonstrated that Pb was mainly complexed to organic acids within plant tissues whereas the dominant form in soil was PbSO4. Parallel to the soil-plant Pb transfer assays, a genetic transformation protocol was optimized in view of better understanding biochemical processes involved in lead hyperaccumulation and gene function, in the future. The best regeneration scheme was based on the pre-culture of explants on 10 μM TDZ (Thidiazuron) in addition to 1 mg L-1 each of N6-benzylaminopurine (BAP) and α- naphthaleneacetic acid (NAA), followed by removal of TDZ from the culture medium. Kanamycin and hygromycin proved to be efficient selectable markers for genetic transformation. Two Agrobacterium strains, C58 and EHA105 harboring binary vectors carrying the selectable marker genes hpt and nptII were chosen for transformation experiments. They also contained the uidA gene coding sequence as reporter gene. After infecting with C58, 4 and 107 rooted plants on hygromycin-containing medium were obtained for Attar and Atomic cultivars, respectively. The four Attar plants and 82 Atomic plants expressed Gus in leaves, petioles, stems and roots as expected with a sequence driven by the 35S constitutive promoter. Polymerase Chain Reaction (PCR) screening was performed on Gus positive plants and 2 and 20 plants of Attar and Atomic were screened as PCR positive, respectively. After infection with EHA105, 23 and 133 rooted plants were obtained on kanamycin selection medium but none of these expressed Gus. Southern hybridization patterns will enable to correlate gene copy numbers to expression levels in these different events. The optimized protocols could be used for understanding molecular mechanisms of Pb accumulation and improvement in phytoextraction technique.