Aspen micropropagation: use for phytoremediation of soils

Toxic pollution of soils is a major environmental problem in the Czech Republic. Most contemporary remediation approaches do not provide acceptable solutions. For environmental clean-up, the use of specially selected and engineered plants capable of effective restoration and stabilization of contaminated sites is an emerging technology called phytoremediation. Aspen (Populus spp.) trees represent optimal plants for absorption, accumulation, storage, and degradation of environmental pollutants such as heavy metals, pesticide residues, and other waste products. The first aim of this study was to verify possibilities of micropropagating selected elite European aspen (Populus tremula) and hybrid aspen (Populus tremula × Populus tremuloides) mature trees growing in polluted areas. The primary cultures were established from 24 years old trees from provenance plots. Dormant buds were sampled from selected trees in spring. After sterilization of dormant buds, the shoot tips were extirpated and put on nutrient media. MS medium with a higher concentration of BAP (1.0 mg/l) and IBA (0.1 mg/l) showed to be suitable for induction of organogenesis. MS medium with lower concentration of BAP (0.2 mg/l) and higher concentration of glutamine (100 mg/l) in agar medium was used for multiplication. A high number of adventitious shoots (20-30) was produced per multi-apex culture. The losses during rooting and acclimatization were minimal, around 2%. Currently, the plantlets grow on the outside bed of the experimental nursery. Twenty-five clones were established in the gene bank. Secondly, standardization of the efficient transformation of hybrid aspen was investigated with the aim to facilitate production of transformed hybrid aspen with valuable genes for increased capability of phytoremediation in the near future. Four gene constructs of Agrobacterium tumefaciens comprising different promoters (bearing two genes, NPTII coding resistance to kanamycin, and GUS coding glucuronidase synthesis allowing histochemical and fluorometrical identification) were used for transformations of aspen hybrid stem segments. A promoter suitable for transformations was selected on the basis of GUS activity assay. The highest activity was estimated in transformants with construct 148 controlled by promoter CAMV 35S.