Root enhancement in cytokinin-deficient oilseed rape causes leaf mineral enrichment, increases the chlorophyll concentration under nutrient limitation and enhances the phytoremediation capacity

Background Cytokinin is a negative regulator of root growth, and a reduction of the cytokinin content or signalling causes the formation a larger root system in model plants, improves their growth under drought and nutrient limitation and causes increased accumulation of elements in the shoot. Roots are an important but understudied target of plant breeding. Here we have therefore explored whether root enhancement by lowering the cytokinin content can also be achieved in oilseed rape (Brassica napus L.) plants. Results Transgenic plants overexpressing the CKX2 gene of Arabidopsis thaliana encoding a cytokinin-degrading cytokinin oxidase/dehydrogenase showed higher CKX activity and a strongly reduced cytokinin content. Cytokinin deficiency led to the formation of a larger root system under different growth conditions, which was mainly due to an increased number of lateral and adventitious roots. In contrast, shoot growth was comparable to wild type, which caused an enhanced root-to-shoot ratio. Transgenic plants accumulated in their leaves higher concentrations of macro- and microelements including P, Ca, Mg, S, Zn, Cu, Mo and Mn. They formed more chlorophyll under Mg- and S-deficiency and accumulated a larger amount of Cd and Zn from contaminated medium and soil. Conclusions These findings demonstrate the usefulness of ectopic CKX gene expression to achieve root enhancement in oilseed rape and underpin the functional relevance of a larger root system. Furthermore, the lack of major developmental consequences on shoot growth in cytokinin-deficient oilseed rape indicates species-specific differences of CKX gene and/or cytokinin action

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

In vitro breeding of Brassica juncea L. to enhance metal accumulation and extraction properties

In vitro breeding and somaclonal variation were used as tools to improve the potential of Indian mustard (Brassica juncea L.) to extract and accumulate toxic metals. Calli from B. juncea were cultivated on a modified MS medium supplemented with 10–200 μM Cd or Pb. Afterwards, new B. juncea somaclones were regenerated from metal-tolerant callus cells. Three different phenotypes with improved tolerance of Cd, Zn and Pb were observed under hydroponic conditions: enhanced metal accumulation in both shoots and roots; limited metal translocation from roots to shoots; reduced accumulation in shoots and roots. Seven out of thirty individual variants showed a significantly higher metal extraction than the control plants. The improvement of metal shoot accumulation of the best regenerant (3× Cd, 1.6× Zn, 1.8× Pb) and metal extraction (6.2× Cd, 3.2× Zn, 3.8× Pb) indicated a successful breeding and selection of B. juncea, which could be used for phytoremediation purpose

Phenotypic seedling responses of a metal-tolerant mutant line of sunflower growing on a Cu-contaminated soil series: potential uses for biomonitoring of Cu exposure and phytoremediation

Background and aims The potential use of a metal-tolerant sunflower mutant line for both biomonitoring and phytoremediating a Cu-contaminated soil series was investigated. Methods The soil series (21–1,170 mg Cu kg−1) was sampled in field plots at control and wood preservation sites. Sunflowers were cultivated 1 month in potted soils under controlled conditions. Results pH and dissolved organic matter influenced Cu concentration in the soil pore water. Leaf chlorophyll content and root growth decreased as Cu exposure rose. Their EC10 values corresponded to 104 and 118 μg Cu L−1 in the soil pore water, 138 and 155 mg Cu kg−1 for total soil Cu, and 16–18 mg Cu kg−1 DW shoot. Biomass of plant organs as well as leaf area, length and asymmetry were well correlated with Cu exposure, contrary to the maximum stem height and leaf water content. Conclusions Physiological parameters were more sensitive to soil Cu exposure than the morphological ones. Bioconcentration and translocation factors and distribution of mineral masses for Cu highlighted this mutant as a secondary Cu accumulator. Free Cu2+ concentration in soil pore water best predicted Cu phytoavailability. The usefulness of this sunflower mutant line for biomonitoring and Cu phytoextraction was discussed

Stability of enhanced yield and metal uptake by sunflower mutants for improved phytoremediation

Plant biomass and metal shoot accumulation are key factors for efficient phytoextraction. In a previous study, chemical mutagenesis has been used to improve the phytoextraction potential of sunflowers. The main goal of the present study was to assess the stability of sunflower mutants with improved biomass and metal accumulation properties in the 3rd and 4th generations. As compared to control plants, the best M3 mutants showed the follwing improvement of metal extraction: Cd 3-5-fold, Zn 4-5-fold, and Pb 3-5-fod. The best M4 sunflowers also shoed enhanced metal extraction: Cd 3-4- fond, Zn 5-7-fold, Pb 6-8-fold and Cr 5-7-fold. The control sunflower inbred line IBL 04, grown directly ion the field, accumulated metals in individual organs in the following decreasing order: Cd and Zn: leaves > stem > roots > flower > seeds; Cr: roots > flower > seeds > leaves > stem. The best sunflower mutants showed either higher metal accumulation in shoots or enhanced metal accumulation in roots, suggesting to improved phytoextraction or rhizofiltration efficiency, respectively

Feasibility Of Labile Zn Phytoextraction Using Enhanced Tobacco And Sunflower: Results Of Five- And One-Year Field-Scale Experiments In Switzerland

Phytoextraction with somaclonal variants of tobacco and sunflower mutant lines (non-GMs) with enhanced metal uptake and tolerance can be a sustainable alternative to conventional destructive decontamination methods, especially for stripping bioavailable zinc excess in topsoil. The overall results of a 5-year time series experiment at field scale in north-eastern Switzerland confirm that the labile Zn pool in soil can be lowered by 45-70%, whereas subplots without phytoextraction treatment maintained labile Zn concentrations. In 2011, the phytoextraction experiment site was enlarged by a factor of 3, and the labile 0.1M NaNO3 extractable Zn concentration in the soil was reduced up to 58% one period after harvest. A Mass Balance Analysis confirmed soil Zn decontamination in line with plant Zn uptake. The plants partially take Zn from the non-labile pool of the total. The sustainability of Zn phytoextraction in subplots that no longer exceed the Swiss trigger value is now assessed over time. In contrary to the phytoextraction of total soil Zn which needs a long cleaning up time, the bioavailable Zn stripping is feasible within a few years period

A Promising Technique to Increase Metal Concentration and Extraction in Sunflowers

Since most of the metal-hyperaccumulating wild plants only produce very low biomass and many high-yielding crops accumulate only moderate amounts of metals, the current research is mainly focused on overcoming these limitations and the optimization of metal phytoextraction. The main goal of the present study was the improvement of metal concentration and extraction properties of Helianthus annuus L by chemical mutagenesis (the non-GMO approach). Sunflowers - hybrid cultivar Salut and inbred lines - were treated with the chemical mutagen ethyl methanesulfonate (EMS). The effect of chemical mutagenesis on metal concentration in and extraction by new sunflower M1 and M2 mutants was directly assessed on a metal-contaminated field in Rafz, Switzerland. Mutants of the M2 generation showed a 2-3 times higher metal shoot concentration than the control plants. The best M2 sunflower "giant mutant" 14/185/04 showed a significantly enhanced metal extraction ability: 7.5 times for Cd, 9.2 times for Zn, and 8.2 times for Pb in aboveground parts, as compared to the control plants. Theoretical calculations for the phytoextraction potential of new sunflower variants note that the best sunflower mutant can produce up to 26 t dry matter per hectare and remove 13.3 kg Zn per hectare and year at the sewage sludge contaminated site of Rafz; that is a gain factor of 9 compared to Zn extraction by sunflower controls. Furthermore, the use of sunflower oil and biomass for technical purposes (lubricants, biodiesel, biogas) should produce an additional value and improve the economical balance of phytoextraction

Root-Specific Reduction of Cytokinin Causes Enhanced Root Growth, Drought Tolerance, and Leaf Mineral Enrichment in Arabidopsis and Tobacco[C][W][OA]

Root-specific expression of a cytokinin-degrading CKX gene induces the formation of a larger root system, whereas growth and development of the shoot remain similar. Transgenic plants exhibiting a larger root system had a higher survival rate after severe drought treatment and an increased leaf element content, suggesting the approach might be useful to optimize crop plants

Screening of sunflower cultivars for metal phytoextraction in a contaminated field prior to mutagenesis

Sunflower can be used for the remediation of metal-contaminated soils. Its high biomass production makes this plant species interesting for phytoextraction and using sunflower oil for a technical purpose may improve the economic balance of phytoremediation. The aim of the present field study was to screen 15 commercial cultivars of Helianthus annuus L., grown on metal-contaminated soil, to find out the variety with the highest metal extraction, which can be further improved by mutation or in vitro breeding procedures. Two different fertilizers (ammonium sulphate and ammonium nitrate) were also used to enhance the bioavailability of metals in soil. Highly significant differences were observed within tested varieties for metal accumulation and extraction efficiency. Furthermore, ammonium nitrate increased cadmium extraction, whereas ammonium sulphate enhanced zinc and lead uptake in most tested cultivars. In this field-based sunflower screening, we found enhanced cumulative Cd, Zn, and Pb extraction efficiency by a factor 4.4 for Salut cultivar. We therefore emphasize that prior to any classical breeding or genetic engineering enhancing metal uptake potential, a careful screening of various genotypes should be done to select the cultivar with the naturally highest metal uptake and to start the genetic improvement with the best available plant material