Non-GMO approach for the improvement of heavy metal accumulation and extraction of high yielding crop species for efficient phytoextraction of contaminated soil

Some plants able to take up heavy metals from contaminated soils offer a possibility to clean up sites contaminated with heavy metals. Plants thus act as a solar driven pump, which can extract and concentrate heavy metals from the environment. Since most of the metal hyperaccumulating wild plants only produce very low biomass and most of plants producing high biomass accumulate only moderate amounts of metals, the current research is mainly focused on the overcoming of this deficiency to optimise metal phytoextraction. The main goal of this EC financed study was aimed at improvement of phytoextraction through improved metal extraction of high yielding oil crops, such as Helianthus annuus L. and Brassica juncea L. producing a high biomass. The use of their oil and biomass for technical purpose (lubricants, biogas and energy) allows to produce an additional value from this in situ decontamination technique and to improve the economical balance of phytoextraction. The enhancement of metal accumulation properties of sunflower and Indian mustard by non-GMO approach and of stimulated metal bioavailability in the soil were the main milestones of the present study. Classical fertilisers were used to lower soil pH, increasing metal availability from the soil and conventional biotechnological approaches were used as an alternative to genetic engineering to enhance both metal accumulation and extraction efficiency of oil crops. The first field experimentation (2002) was mainly focused on the screening of 15 commercial sunflower cultivars growing on a contaminated field in Rafz (Switzerland) to select the cultivar with the naturally highest potential to accumulate and extract metals from contaminated soil, and to assess the effect of classical fertilisers on metal accumulation/extraction of the sunflower cultivars. Highly significant differences of heavy metal accumulation and extraction were found between cultivars. Cadmium extraction varied by a factor of 4, Zn extraction by factor of 3 and Pb extraction by a factor of 14 between the cultivars with the highest and lowest metal extraction treated with the same fertiliser. Sulphate fertilisation significantly enhanced Zn and Pb extraction, whereas ammonium nitrate enhanced Cd extraction by most of the sunflower cultivars. Cultivar Salut showed the highest Cd, Zn and Pb extraction and was chosen for the next mutation breeding. Classical mutation breeding techniques were assumed to be efficient to improve the efficiency of metal accumulation of high yielding crops. Therefore, in vitro breeding was used to improve metal uptake of Indian mustard and chemical mutagenesis using ethyl methanesulphonate (EMS) was additionally applied for sunflowers, hybrid cultivar Salut and genetically homogenous inbred lines. Somaclonal variation of tissue culture was used as a source of genetic variability to increase the potential of metal accumulation in Indian mustard. After the selection of B. juncea callus cultures on a medium spiked with 10-200 μM of Cd or Pb, new somaclonal variants were regenerated from metal tolerant callus cells. A subsequent screening of 30 new B. juncea regenerants growing on a hydroponic medium spiked with toxic metals showed that 7 new somaclones (23 %) possessed a significantly higher shoot metal extraction than the control plants. The best regenerant showed a 6 times higher Cd, a 3 times higher Zn and a 4 times higher Pb extraction in the shoots than the control. Prior to the mutation breeding of sunflower, the effects of toxic metals on the growth, metal accumulation in shoot and root, metal translocation of control sunflower cultivars were investigated on a hydroponic medium spiked with toxic metal. Based on phytotoxic effects of roots and shoots the assessed sunflowers showed the following tolerance towards toxic metals: Pb >> Zn > Cd. The effect of the chemical mutagenesis on the efficiency of metal accumulation and extraction was first assessed on a hydroponic system, spiked with Cd, Zn and Pb. The results show that the EMS mutagenesis affected metal uptake, root and shoot metal accumulation and root to shoot metal translocation in mutant variants. We obtained M1 sunflower mutants with an enhanced metal accumulation, mutants with a lower metal accumulation and mutants without changed metal accumulation characteristics compared to the control plants. The next two field experiments in 2003 and 2004 were focused on the screening of 500 sunflower mutants of M1 generation and 300 sunflower mutants of M2 generation on the metal contaminated Rafz site to assess the effect of mutagenesis on yield, metal accumulation and extraction. The M1 sunflower mutants showed a 2-3 times higher Cd, Zn and Pb concentration in shoots, but a considerably reduced growth, as compared to control cultivars due to the phytotoxic effect of the mutagen. The sunflower mutants of M2 generation also showed a 2-3 times higher metal shoot accumulation than the control plants, and even more the metal extraction of the best M2 sunflower mutants "Giant Mutant 14/190/04" was increased 7.5 times for Cd, 8.2 times for Zn and 9.2 times for Pb extraction compared to the control plants. Theoretical calculations of phytoextraction potential of sunflower point out that the best sunflower mutant can produce up to 26 t dry matter yield per ha and remove 13.3 kg Zn per ha and year at the metal contaminated site in Rafz (Switzerland), that is a gain of factor 9 compared to Zn removal of control sunflowers. From a practical point of view this improvement looks very promising for boosting future phytoextraction research