super plants: is phytoremediation the solution to heavy

Super plants: is phytoremediation the solution to heavy metal contamination?

Required reading Bhargava, A., Carmona, F.C., Bhargava, M., & Srivastava, S., 'Review: Approaches for enhanced phytoextraction of heavy metals', Journal Of Environmental Management, 105, pp. 103-120. General discussion points

• Should we be worried about releasing genetically modified plants (for

phytoextraction) into the environment?

• How green/environmentally friendly an approach is phytoremeditation

in comparison with other remediation methods?

• What can you do to reduce soil contamination? (Brainstorm 3

actionable ideas and go do one!)

Questions

1. How does phytoextraction work?

2. What are some of the factors affecting heavy metal uptake by plants?

3. How could plants be enhanced to increase heavy metal

phytoextraction?

4. What are the limitations to phytoextraction of heavy metals?

5. Is phytoextraction of heavy metals commercially viable? What are its

prospects for the future?

Bhargava, A., Carmona, F.C., Bhargava, M., & Srivastava, S., 'Review: Approaches for enhanced phytoextraction of heavy metals', Journal Of Environmental Management, 105, pp. 103-120.

Review

Approaches for enhanced phytoextraction of heavy metals

Atul Bhargava a,*, Francisco F. Carmona b, Meenakshi Bhargava c,1, Shilpi Srivastava a

aAmity Institute of Biotechnology, Amity University Uttar Pradesh, Lucknow Campus, Gomti Nagar, Lucknow, UP 226010, IndiabDepartamento de Agricultura del Desierto y Biotecnología, Universidad de Arturo Prat, Avenida Arturo Prat 2120, Campus Huayquique, Iquique, ChilecDepartment of Chemistry, University of Allahabad, Allahabad, India

a r t i c l e i n f o

Article history:Received 27 June 2011Received in revised form20 March 2012Accepted 1 April 2012Available online 27 April 2012

Keywords:PhytoextractionHeavy metalsBiochemical mechanismsCation transportersMetallothioneinsPhytochelatinsPlant improvementTransgenics

a b s t r a c t

The contamination of the environment with toxic metals has become a worldwide problem. Metaltoxicity affects crop yields, soil biomass and fertility. Soils polluted with heavy metals pose a serioushealth hazard to humans as well as plants and animals, and often requires soil remediation practices.Phytoextraction refers to the uptake of contaminants from soil or water by plant roots and their trans-location to any harvestable plant part. Phytoextraction has the potential to remove contaminants andpromote long-term cleanup of soil or wastewater. The success of phytoextraction as a potential envi-ronmental cleanup technology depends on factors like metal availability for uptake, as well as plantsability to absorb and accumulate metals in aerial parts. Efforts are ongoing to understand the geneticsand biochemistry of metal uptake, transport and storage in hyperaccumulator plants so as to be able todevelop transgenic plants with improved phytoremediation capability. Many plant species are beinginvestigated to determine their usefulness for phytoextraction, especially high biomass crops. Thepresent review aims to give an updated version of information available with respect to metal toleranceand accumulation mechanisms in plants, as well as on the environmental and genetic factors affectingheavy metal uptake. The genetic tools of classical breeding and genetic engineering have opened thedoor to creation of ‘remediation’ cultivars. An overview is presented on the possible strategies fordeveloping novel genotypes with increased metal accumulation and tolerance to toxicity.

! 2012 Elsevier Ltd. All rights reserved.

1. Introduction

The rising rate of human activities in the biosphere is posingunprecedented threats that would ultimately lead to a disturbingimbalance in the biosphere. Industrial activities such as chemicalworks, service stations, metal fabrication shops, paper mills,tanneries, textile plants, waste disposal sites and intensive agri-culture are particularly guilty of polluting the environment(Wong, 2003; Freitas et al., 2004). Heavy metal contamination ofsoils has become a serious problem in areas of intense industryand agriculture. A heavy metal is a member of an ill-definedsubset of elements that exhibit metallic properties, which mainlyincludes the transition metals, some metalloids, lanthanides andactinides (Babula et al., 2008). These are metallic chemicalelements that have a relatively high density and are toxic even atlow concentrations. The contamination of the environment withheavy metals has become a worldwide problem that affects crop

yields, soil biomass and fertility, and leads to bioaccumulation ofmetals in the food chain (Gratao et al., 2005; Rajkumar et al.,2009). This is majorly due to pollution of agricultural soils byincreasing reliance on chemical fertilizers, which has imposeda long-term risk on environmental health (McLaughlin et al., 1999;Wong et al., 2002). Industrialized countries have regulated theemission of toxic substances, but in developing countries, rapidindustrial development and population explosion, coupled withlack of pollution control has caused an enormous increase inheavy metal contamination of agricultural soils (Ji et al., 2000).Soils polluted with heavy metals pose a health hazard to humansas well as plants and animals. Thus, heavy metals need to beremoved from the soil for agro-ecological sustainability andhuman benefit.

Various efficient soil cleanup techniques are available, butmost of them are costly, labour intensive and cause soil distur-bances, and have thus found limited acceptability among thecommunities. Conventional remediation methods usually involvepneumatic fracturing, solidification/stabilization, vitrification,excavation and removal of contaminated soil layer, physicalstabilization or washing of contaminated soils with strong acids orheavy metal chelators (Steele and Pichtel, 1998; Khan et al., 2004;

* Corresponding author. Tel.: þ91 522 2721931/32; fax: þ91 522 2721934.E-mail address: [email protected] (A. Bhargava).

1 Present address: Delhi Public School, Indira Nagar, Lucknow, India.

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Bhargava et al., 2012). Phytoremediation, using plants to cleanupcontaminated environment, is an idea that is attracting increasingattention among scientists, remediation engineers, and environ-mental professionals in government and industries. In situremediation using plants has the potential to be less expensivethan current technologies and simultaneously initiates bothdetoxification of hazardous waste and site restoration. Phytor-emediation technologies include phytoextraction, phytostabili-zation, phytovolatilization, phytofiltration and phytodegradation(Fulekar et al., 2009; Marques et al., 2009). Phytoextractionrefers to the uptake of contaminants from soil or water by plantroots and their translocation into the shoot, or any otherharvestable plant part, to remove contaminants and promotelong-term cleanup of soil or wastewater (Sas-Nowosielska et al.,2008). In this approach, plants capable of accumulating heavymetals are grown on contaminated sites and the metal-richaboveground biomass is harvested on maturity. As a result,a fraction of the soil contaminant is removed. The success ofphytoextraction as a potential environmental cleanup technologydepends on factors like metal availability for uptake as well asplant ability to absorb and accumulate metals in its aerial parts. Asper the economic feasibility, the harvested biomass is usuallyincinerated or composted and rarely recycled for reuse (Prasadand Freitas, 2003).

Plants ideal for phytoextraction should possess multiple traitslike ability to grow outside their area of collection, fast growth, highbiomass, easy harvesting and accumulation of a range of heavymetals in their harvestable parts (Jabeen et al., 2009; Seth, 2011).No plant is presently known that fulfils all these criteria. However,a rapidly growing non-accumulator plant could be modified and/orengineered so that it achieves most of the above-mentioned attri-butes. There has been significant progress in determining thebiochemical and molecular basis for metal accumulation, whichprovides us with a strong scientific basis to outline some strategiesfor achieving this goal. This article first aims to review studies thatare beginning to uncover the detailed mechanism behind phy-toextraction of heavy metals in plants. The progress of researchfocused on the unravelling of genetic and biochemical mechanismsthat confer the ability to accumulate heavy metals into plants isdescribed. Plant breeders and environmental researchers have longstrived to develop improved plant varieties which can be used foreffective phytoextraction. This aspect is been discussed in thisreview, and the use of classical and biotechnological approachesused for enhancing natural hyperaccumulation of heavy metals ispresented.

2. Plants as accumulators of heavy metals

The uptake of metals by plants is selective, with some beingpreferentially acquired over others. Many heavy metals like nickel(Ni), copper (Cu), manganese (Mn) and zinc (Zn) are essentialmicronutrients and required by plants to grow and complete thelife cycle. According to Baker (1981), plants growing on metallif-erous soils can be grouped into three categories: (i) Excluders e inwhich the metal concentrations in the shoot are maintained up toa critical value, at a low level across a wide range of soil concen-tration. Excluders prevent uptake of toxic metals into root cells(de Vos et al., 1991). Excluders can be used for stabilization of soiland to avoid further spread of contamination due to erosion (Lasat,2002). (ii) Accumulators e in which the metals are concentrated inaboveground plant parts from low to high soil concentrations.Accumulators do not prevent metals from entering the roots andthus allow bioaccumulation of high concentration of metals.(iii) Indicators e where internal concentration reflects the externallevels (McGrath et al., 2002).

2.1. Hyperaccumulators

The discovery of hyperaccumulator plant species has revolu-tionized phytomediation technology since these plants have aninnate capacity to absorbmetal at levels 50e500 times greater thanaverage plants (Lasat, 2000). Hyperaccumulators are a subgroup ofaccumulator species often endemic to naturally mineralized soils,which accumulate high concentrations of metals in their foliage(Baker and Brooks, 1989; Raskin et al., 1997). Metal hyper-accumulators are naturally capable of accumulating heavy metalsin their aboveground tissues, without developing any toxicitysymptoms. A metal hyperaccumulator is a plant that can concen-trate the metals to a level of 0.1% (of the leaf dry weight) for Ni, Co,Cr, Cu, Al and Pb; 1% for Zn and Mn; and 0.01% for Cd and Se (Bakerand Brooks, 1989; Baker et al., 2000). The time taken by plants toreduce the amount of heavy metals in contaminated soils dependson biomass production as well as on their bioconcentration factor(BCF), which is the ratio of metal concentration in the shoot tissueto the soil (McGrath and Zhao, 2003). It is determined by thecapacity of the roots to take up metals and their ability to accu-mulate, store and detoxify metals while maintaining metabolism,growth and biomass production (Gleba et al., 1999; Guerinot andSalt, 2001; Clemens et al., 2002). With the exception of hyper-accumulators, most plants have metal bioconcentration factors ofless than 1, which means that it takes longer than a human lifespanto reduce soil contamination by 50% (Peuke and Rennenberg,2005). Hyperaccumulators have a bioconcentration factor greaterthan 1, sometimes reaching as high as 50e100. The relationshipbetween metal hyperaccumulation and tolerance is still a subjectof debate. Views range from no correlation between hyper-accumulators and the degree of tolerance to metals (Baker et al.,1994) to strong association between these traits (Chaney et al.,1997). It is increasingly being realized that to cope with highconcentrations of metals in their tissue, plants must also toleratethe metals that they accumulate.

There has long been a general agreement that metal hyper-accumulation is an evolutionary adaptation by specialized plants tolive in habitats that are naturally rich in specific minerals thatconfers on them the qualities of increased metal tolerance,protection against herbivores or pathogens, drought tolerance, andallelopathy (Boyd and Martens, 1992; Macnair, 1993). The hypoth-esis of protection against pathogens and herbivores is consideredthe most accepted one (Boyd and Martens, 1994; Huitson andMacnair, 2003; Boyd, 2007; Noret et al., 2007; Galeas et al.,2008). However, the mechanisms of metal uptake, tolerance tohigh metal concentrations, and the exact roles that high level ofelements play in the survival of hyperaccumulators have continuedto be debated.

Hyperaccumulation of heavy metal ions is a striking phenom-enon exhibited by approximately <0.2% of angiosperms (Baker andWhiting, 2002; Rascio and Navari-Izzo, 2011). Metal hyper-accumulators have been reported to occur in over 450 species ofvascular plants from 45 angiosperm families (Table 1) includingmembers of the Asteraceae, Brassicaceae, Caryophyllaceae, Cyper-aceae, Cunoniaceae, Fabaceae, Flacourtiaceae, Lamiaceae, Poaceae,Violaceae, and Euphorbiaceae (Padmavathiamma and Li, 2007), butare well represented in Brassicaceae especially in the generaAlyssum and Thlaspi, wherein accumulation of more than one metalhas been reported (Reeves and Baker, 2000; Prasad and Freitas,2003; Verbruggen et al., 2009; Vamerali et al., 2010) (Table 1).Pteris vittata (Chinese brake fern) is known to accumulate up to 95%of the arsenic taken up from soil in its fronds (Ma et al., 2001; Zhanget al., 2002). The best known angiosperm hyperaccumulator ofmetals is Thlaspi (now: Noccaea) caerulescens (pennycress), whichcan accumulate large amounts of Zn (39,600 mg/kg) and Cd

A. Bhargava et al. / Journal of Environmental Management 105 (2012) 103e120104

(1800 mg/kg) without apparent damage (Brown et al., 1995; Basicet al., 2006; Rascio and Navari-Izzo, 2011). This small, self-pollinating diploid plant can easily grow under lab conditions andtherefore represents an excellent experimental system for studyingthe mechanisms of metal uptake, accumulation and tolerance inrelation to metal phytoextraction. Apart from T. caerulescens,Brassica juncea has also been used as a model system to investigatethe physiology and biochemistry of metal accumulation in plants.

2.2. High biomass crops

For successful and economically feasible phytoextraction, it isnecessary to use plants having ametal bioconcentration factor of 20and a biomass production of 10 tonnes per hectare (t/ha); or plantswith a metal bioconcentration factor of 10 and a biomass produc-tion of 20 t/ha (Peuke and Rennenberg, 2005). The rate of phy-toextraction is directly proportional to plant growth rate and thetotal amount of metal phytoextracted is correlated with the plantbiomass, which makes the process of phytoextraction very slow(Shah and Nongkynrih, 2007). This necessitates the identification offast growing (largest potential biomass and greatest nutrientresponses) and strongly metal-accumulating genotypes. B. juncea,while having one-third the concentration of Zn in its tissue, isconsidered to be more effective at Zn removal from soil thanT. caerulescens, a known hyperaccumulator of Zn (Ebbs et al., 1997).

This advantage is primarily due to the fact that B. juncea producesten-times more biomass than T. caerulescens.

Recently, interest has arisen in the use of high-biomass crops forphytoextraction of metals (Doty, 2008; Capuana, 2011). Fast-growing trees are ideal low cost candidates for phytoextractiondue to their extensive root systems, high rates of water uptake andtranspiration, rapid growth, large biomass production and easyharvesting with subsequent resproutingwithout disturbance of thesite (Peuke and Rennenberg, 2005). Several tree species haveevoked interest in the phytoremediation of metal contaminatedsoils and show great prospects for heavy metal remediation(Pulford andWatson, 2003; Rosselli et al., 2003; Meers et al., 2007;Unterbrunner et al., 2007; Brunner et al., 2008; Domínguez et al.,2008). Poplar and willow, though not hyperaccumulators, areeffective because of their greater biomass and deep root systems,which makes them effective remediators of metal contamination.Poplars can be grown in a wide range of climatic conditions and areused with increasing frequency in ‘short-rotation forestry’ systemsfor pulp and paper production (Peuke and Rennenberg, 2005). Thisraises the possibility of using plantations of poplars across severalmultiyear cycles to remove heavy metals from contaminated soils.Importantly, it is unlikely that poplars will enter the human foodchain or end up as feedstock for animals. Likewise, several speciesof willow (Salix dasyclados, Salix smithiana and Salix caprea) displaygood accumulation capabilities and remediation effectiveness,

Table 1Important plant species that are metal hyperaccumulators.

Metal Number of hyperaccumulatorspecies reported

Plant species that accumulatespecific metals

Family Reference

Ni 320 Berkheya coddii Asteraceae Robinson et al. (1997); Moradi et al. (2010)Alyssum serpyllifolium, A. bertolonii Brassicaceae Becerra-Castro et al. (2009); Barzanti et al. (2011)Sebertia acuminata Sapotaceae Jaffre et al. (1976); Perrier (2004)Phidiasia lindavii Acanthaceae Reeves et al. (1999)Bornmuellera kiyakii Brassicaceae Reeves et al. (2009)

Cu 34 Ipomea alpina Convolvulaceae Cunningham and Ow (1996)Crassula helmsii Crassulaceae Küpper et al. (2009)Commelina communis Commelinaceae Wang and Zhong (2011)

Co 34 Haumaniastrum robertii Lamiaceae Brooks (1998)Crotalaria cobalticola Fabaceae Oven et al. (2002)

Se 20 Astragalus bisulcatus Fabaceae Galeas et al. (2007)Stanleya pinnata Brassicaceae Freeman et al. (2010); Hladun et al. (2011)

Zn 18 Thlaspi caerulescens Brassicaceae Kupper and Kochian (2010)Arabis gemmifera, A. paniculata Brassicaceae Kubota and Takenaka (2003); Tang et al. (2009)Sedum alfredii Crassulaceae Sun et al. (2005)Arabidopsis halleri Brassicaceae Zhao et al. (2000)Picris divaricata Asteraceae Du et al. (2011)

Pb 14 Sesbania drummondii Fabaceae Sahi et al. (2002); Sharma et al. (2004)Hemidesmus indicus Apocynaceae Chandra Sekhar et al. (2005)Arabis paniculata Brassicaceae Tang et al. (2009)Plantago orbignyana Plantaginaceae Bech et al. (2011)

Mn 9 Austromyrtus bidwillii Myrtaceae Bidwell et al. (2002)Phytolacca americana Phytolaccaceae Pollard et al. (2009)Virotia neurophylla Proteaceae Fernando et al. (2006)Gossia bidwillii Myrtaceae Fernando et al. (2007)Maytenus founieri Celastraceae Fernando et al. (2008)

Cd 04 Thlaspi caerulescens Brassicaceae Basic et al. (2006)Arabidopsis halleri Brassicaceae Dahmani-Muller et al. (2000); Bert et al. (2002)Bidens pilosa Asteraceae Sun et al. (2009)

Cr naa Salsola kali Amaranthaceae Gardea-Torresday et al. (2005)Leersia hexandra Poaceae Zhang et al. (2007)Gynura pseudochina Asteraceae Mongkhonsin et al. (2011)

Tl naa Iberis intermedia Brassicaceae Leblanc et al. (1999)Brassica oleracea Brassicaceae Al-Najar et al. (2005)

a Not available.

A. Bhargava et al. / Journal of Environmental Management 105 (2012) 103e120 105

similar to herbaceous hyperaccumulators like Arabidopsis halleriand T. caerulescens, compensating lower metal content in shootswith higher biomass production (Fischerová et al., 2006; Kelleret al., 2006; Meers et al., 2007). However, the use of perennialtree species having extensive root systems with elevated metalcontent would require excavation and disposal, especially short-rotation coppice (SRC) after several harvests and at the processend (Mench et al., 2010).

3. Endophytes and phytoextraction

In the last few years, a lot of research has brought forward therole of endophytic bacteria in phytoextraction of pollutants (Doty,2008). Endophytes are the microbes that inhabit the internal ofplant tissues without causing harm to the host (Kuklinsky-Sobralet al., 2004). Endophytes can facilitate plant growth, increaseresistance of plants to pathogens, drought and herbivores (Bottiniet al., 2004; Saikkonen et al., 2006; Taghavi et al., 2010). Till nowmost of the studies have focused on biodegradation of organicpollutants and applications of endophytic bacteria for improvingphytoremediation of heavy metals. This has been slow because oflack of valuable strains having the heavy metal resistance anddetoxification capacities (Luo et al., 2011).

Although most of the research on endophytes that assist inphytoremediation has focused on bacteria, the arbuscular mycor-rhizal (AM) fungi are also involved in uptake of elements into plants(Doty, 2008). AM fungi are soil-borne obligate biotrophs that areintegral functioning parts of plant living in mutualistic associationwith the roots of about 80% terrestrial plants (Smith and Read,2008). AM fungi are also reported to be present on the roots ofplants growing on heavy metal-contaminated soils and play animportant role in metal accumulation and tolerance (Gaur andAdholeya, 2004; Khade and Adholeya, 2009; Javaid, 2011;Miransari, 2011). An important point about treating polluted soilwith mycorrhizal plants is the selection of appropriate AM species.The species selected from areas polluted with heavy metals are themost efficient species which have attained the ability to surviveunder metal stress conditions and hence can act more efficientlyrelative to the other AM species (Miransari, 2011). Mycorrhizalfungi improve phytoextraction bymakingmetals more available foruptake by plants. Improved phytoextraction following mycorrh-ization may be achieved by several mechanisms like increasedtolerance of plants to metals, better plant growth, increasedbiomass production and greater metal concentrations in planttissues (Vamerali et al., 2010). Generally, species of genus Glomusare predominant in the rhizosphere of plants growing in heavymetal-contaminated soils (Khade and Adholeya, 2009; Bedini et al.,2010). However, the genotypic variation makes it difficult to iden-tify suitable AM fungi for the restoration of metal-contaminatedsoils. Therefore, sustained efforts are required to identify heavymetal-tolerant mycorrhizal strains for their ultimate application inthe management of metal-contaminated soils (Javaid, 2011).

4. Biochemical mechanisms in accumulators

Plants showing tolerance to toxic metals have a range ofmechanisms at the cellular and molecular level that might beinvolved in the general homeostasis, detoxification and tolerance toheavy metal stress (Hall, 2002). Four processes are generallybelieved to be crucial for accumulation: uptake of metals by roots,transport of metals from roots to shoot, complexation withchelating molecules and compartmentalization into the vacuole(Hall, 2002; McGrath and Zhao, 2003). For effective phytoex-traction, metals must not only be taken up rapidly, but should alsobe transported from the roots to aerial parts of the plant. Despite

the presence of large amounts of metals in the soil, the uptake ofmetals is mainly influenced by their bioavailable fraction (Vameraliet al., 2010). Though abundant in nature, the actual bioavailabilityof some metals is limited because of low solubility of metals inoxygenated water and strong binding to soil particles. Metalavailability and mobility of metals in the rhizosphere is also influ-enced by rhizospheric microbes as well as the root exudates. Withthe exception of Fe, little is known about active mobilization oftrace elements by plant roots. Acidification of the rhizosphere,exudation of carboxylates and mechanisms assisting in the acqui-sition of phosphorus contribute to increasing the bioavailability ofcertainmicronutrients (Chaney et al., 2007). It is assumed thatmostof the accumulated metals are bound to ligands like organic acids,amino acids, peptides and proteins (Verbruggen et al., 2009). Whilesome aspects of metal detoxification by ligands have been discov-ered, there is no complete picture of the different chelatorsinvolved in different stages of the internal transport of metals in theplant and storage in accumulators. There is no conclusive evidencethat hyperaccumulators exude specific chelators in the rhizosphereto enhancemetal uptake. Therefore, the release of specific chelatorsassociated with enhanced metal uptake and translocation needsmore extensive research (do Nascimento and Xing, 2006).

Several plants are known to possess highly specialized mecha-nisms to stimulate metal bioavailability in the rhizosphere formetal uptake. Siderophores are small molecular mass organiccompounds produced by microorganisms and members of familyPoaceae that are capable of enhancing the availability of iron foruptake into roots (Neubauer et al., 2000; Devez et al., 2009).Siderophores are specific Fe (III) ligands which form stablecomplexes with metals like Cd, Cu, Ni, Pb and Zn (Nair et al., 2007).The specific chelation of metals can be of great relevance todecontamination of soil having high metal content. It was sug-gested that transgenic plants could be developed to secrete metalselective ligands into the rhizosphere, which could specificallysolubilize elements of interest (Raskin,1996; Neubauer et al., 2000).The efficiency of phytoextraction may be increased by growingsiderophore producing grass species in combination with accu-mulator plants. Although this approach holds promise, phytosi-derophores obtain their specificity not by chelation specifically onlyof Fe in soils, but from their uptake of Fe-phytosiderophores bya membrane carrier (Parker et al., 1995; Yehuda et al., 1996;Neubauer et al., 2000). Researchers feel that finding other biosyn-thetic molecules with selective chelation ability, which plants canmake and secrete into the rhizosphere at adequate concentrationsand create a selective transport protein for the metal chelate seemsdifficult, but worth examination to develop unique phytor-emediation tools. Attempts have been made to identify ligands,which were believed to be secreted by roots of hyperaccumulatorsto increase the rate of Ni release from soil and/or uptake by roots(Kramer et al., 2000; Pinel et al., 2003; Puschenreiter et al., 2003;Wenzel et al., 2003a, 2003b). One such study compared thehyperaccumulator Thlaspi with wheat species that secreted phy-tosiderophores. The wheat rhizosphere solution containedsubstantial levels of ligand(s) as compared to Thlaspi rhizospheresolution that contained very little (Zhao et al., 2001). Severalresearches support the model in which up-regulation or constitu-tive high activity of element transporters in plasma membranesallow plants to achieve hyperaccumulation (as opposed to usingsecreted ligands) (Chaney et al., 2007). While ligand secretion andother common root processes do not allow plants to achievehyperaccumulation, they can alter elemental speciation inthe rhizosphere and thereby influence the phytoavailability ofelements in the rhizosphere. Although the concept of makingplants secreting chelating ligands into the rhizosphere and thenabsorbing the metaleligand complex into roots remains plausible


for phytoextraction, there is no evidence that such an approachcould be developed into practical phytoextraction systems (Chaneyet al., 2007).

Apart from these natural mechanisms, addition of syntheticchelates is known to stimulate the release of metals into soil solu-tion and therefore enhance the potential for uptake into roots (Lasat,2002). Several chelating agents such as ethylenediaminetetraaceticacid (EDTA), trans-1, 2-cyclohexylenedinitrilotetraacetic acid(CDTA), diethylenetriamine-pentaacetic acid (DTPA), ethyleneglycol-O,O0-bis-[2-amino-ethyl]-N,N,N0,N0, tetracetic acid (EGTA),ethylenediamine di-o-hydroxyphenlyacetic acid (EDDHA), methyl-glycinediacetate (MGDA), nitrilotriacetic acid (NTA) and citric acidhave been studied for their ability to mobilize metals and increasemetal accumulation in plant species. EDTA is most widely used inphytoextraction research and has been successfully utilized toremediate heavy metal contaminated soils (Chen et al., 2004). Theextent of heavy metal solubilization by chelation with organiccomplexing agents follows the order of their stability constants,which are determined in aqueous solutions using the ratio of metalto chelating agent of 1:1. However despite the success of thistechnology, concerns have been raised regarding the enhancedmobility of metals in soil and their potential risk of leaching toground water (Cooper et al., 1999). In addition to the risk of metalleaching, the use of chelators is also limited by their high cost.Chelating agents which are more rapidly degraded by soil microbesyet chelate metals well (eg. EDDS) are more expensive and do notappreciably decrease metal leaching from treated soil (Meers et al.,2005). Chaney et al. (2002) estimated that the cost for the amount ofEDTA reportedly needed to attain over 10 g Pb kg"1 dry shootswould be about $30,000 ha"1 (10 mmol EDTA kg"1 soil for eachcropping).

Following mobilization, metals are first bound to the cell wall,which is an ion exchanger of comparatively low selectivity. Trans-port systems and intracellular high-affinity binding sites thenmediate and drive uptake across the plasma membrane through

secondary transporters such as channel proteins and/or Hþ-coupled carrier proteins (Chaney et al., 2007). A number ofimportant membrane transporter gene families have been identi-fied and characterized by heterologous complementation screensand sequencing of ESTs and plant genomes (Table 2). Several cationtransporters have been identified in recent years most of which arein the ZIP (ZRT, IRT-like protein), nramp (natural resistance-associated macrophage protein), ysl (Yellow-stripe-like trans-porter), nas (Nicotinamine synthase), sams (S-adenosyl-methioninesynthetase), fer (Ferritin Fe (III) binding), cdf (cation diffusionfacilitator), hma (heavy metal ATPase) and ireg (Iron regulatedtransporter) family (Guerinot, 2000; Williams et al., 2000; Talkeet al., 2006; van de Mortel et al., 2006; Kramer et al., 2007;Memon and Schroder, 2009; Maestri et al., 2010) (Table 2).

Once inside the plant, further movement of metal containingsap from roots to the aerial parts is controlled by root pressure aswell as the transpirational pull (Robinson et al., 2003). Metaltransport to the shoot primarily takes place through the xylem.Efficient translocation of metal ions to the shoot requires radialsymplastic passage and active loading into the xylem (Clemens,2006; Xing et al., 2008). Due to extreme toxicity of metals athigh intracellular concentrations, plants catalyze redox reactionsand alter the chemistry of these metal ions thereby allowingtheir accumulation in non-toxic forms. Typical examples includereduction of Cr6þ to Cr3þ in Eichornia crassipes (Lytle et al., 1998)and As5þ to As3þ in B. juncea (Pickering et al., 2000). However, someof the heavy metals like Pb, Zn and Cd do not occur in differentoxidation states. In some cases, intracellular metal is detoxified viabinding to low molecular mass organic compounds, localization inthe vacuoles as a metal-organic acid complex or by binding tohistidine (Persans et al., 1999; Kramer et al., 2000; Rascio andNavari-Izzo, 2011). For some metals like Zn, various mechanismsfor regulation of cytoplasmic metal concentration have been putforward that include sequestration in a subcellular organelle,complexation to low molecular mass organic ligands, low uptake

Table 2Important metal transporter genes in different plant species involved in heavy metal tolerance and accumulation.

Family Gene Plant Metal transported Reference

Zn-regulated transporter (ZRT) zip1-12 Arabidopsis thaliana Zn Weber et al. (2004); Roosens et al. (2008a,b)zip4 Oryza sativa Zn Ishimaru et al. (2005)zip Medicago truncatula Zn Lopez-Millan et al. (2004)znt1-2 T. caerulescens Zn van de Mortel et al. (2006)

Fe-regulated transporter (IRT) irt1 Arabidopsis thaliana Fe Kerkeb et al. (2008)irt1-2 Lycopersicon esculentum Fe Bereczky et al. (2003)irt1-2 T. caerulescens Fe Schikora et al. (2006); Plaza et al. (2007)

Natural resistance-associated macrophageproteins (NRAMP)

nramp1-3 Lycopersicon esculentum Fe Bereczky et al. (2003)nramp4 Thlaspi japonicum Fe Mizuno et al. (2005)nramp1 Malus baccata Fe Xiao et al. (2008)

Cation diffusion facilitator (CDF) mtp1 Arabidopsis thaliana Zn Kawachi et al. (2008)mtp1 Arabidopsis halleri Zn Willems et al. (2007)mtp1 Thlaspi goesingense Zn, Ni Kim et al. (2004)mtp1 Nicotiana tabacum Zn, Co Shingu et al. (2005)

Al activated malate transporter (ALMT) almt1 Triticum sp. Al Sasaki et al. (2004)almt1 Secale cereale Al Collins et al. (2008)

P-Type, ATPase (Heavy Metal Associated) hma8 Glycine max Cu Bernal et al. (2007)hma9 Oryza sativa Cu, Zn, Cd Lee et al. (2007)hma4 Arabidopsis halleri Cd Courbot et al. (2007)hma3 Arabidopsis thaliana Co, Zn, Cd, Pb Morel et al. (2008)

Nicotianamine synthase (NAS) nas2, nas3 Arabidopsis halleri Zn Talke et al. (2006)

Copper transporter copt1 Arabidopsis thaliana Cu Sancenon et al. (2004)Andres-Colas et al. (2010)

Yellow Stripe Like (YSL) ysl2 Arabidopsis thaliana Fe, Cu DiDonato et al. (2004)ysl3 T. caerulescens Fe, Ni Gendre et al. (2006)


across the plasma membrane, precipitation as insoluble salts andactive extrusion across the plasma membrane into the apoplast(Brune et al., 1994).

Chelation and sequestration of metals by particular ligands areimportant mechanisms used by plants to deal with metal stress.The two best-characterized metal-binding ligands in plant cells aremetallothioneins (MTs) (Table 3) and phytochelatins (PCs) (Cobbett,2000; Cobbett and Goldsbrough, 2002). These ligands are widelydistributed in plants, and many physiological and genetic studiesindicate that PCs and MTs are critical for metal tolerance andaccumulation in plants (Hartley-Whitaker et al., 2001; van Hoofet al., 2001; Cobbett and Goldsbrough, 2002). Both form stablecomplexes with metals in the cytosol which can be later seques-tered into the vacuole (Goldsbrough, 2000). PCs, a family of thiol-rich peptides, consists of repetitions of the g-Glu-Cys dipeptidefollowed by a terminal Gly with the basic structure (g-Glu-Cys)n-Gly [(PC)n] where n is in the range of two to five (Memon andSchroder, 2009). Overproduction of PCs appears to be an induc-ible rather than a constitutive mechanism, observed especially inmetal non-tolerant plants as part of their defence mechanismagainst metals (Freeman et al., 2005). Phytochelatins are alsoreported to be involved in arsenic detoxification (Schmoger et al.,2000). MTs are low molecular mass cysteine rich proteins thatwere originally isolated as Cu, Zn and Cd binding proteins inanimals (Gratao et al., 2005; Yang et al., 2009). MTs from plant andanimal sources are being explored for introduction in plants forreducing metal accumulation in shoots by trapping the metal inroots (Table 3). The overexpression of MTs can increase planttolerance to specific metals. A wide range of MT genes from variousorganisms like mouse, hamster, humans, yeast, cyanobacteria andplants have been overexpressed in plants (Table 3). However, theevaluation of transgenic plants expressing mammalian genes hasbeen limited to laboratory assessments only.

The vacuole is generally considered to be the main storage sitefor metals in plant cells especially Cd and Zn. Compartmentaliza-tion of metals in the vacuole is an important part of the tolerancemechanism of some metal hyperaccumulator plants. The Nihyperaccumulator Thlaspi goesingense enhances its Ni tolerance bycompartmentalizing most of the intracellular leaf Ni into thevacuole (Kramer et al., 2000). High level of metal ion transporterTgMTP1 in T. goesingense is considered to be the main factorresponsible for the enhanced ability to accumulate metal ionswithin shoot vacuoles (Persans et al., 2001). Intact vacuoles isolatedfrom tobacco and barley exposed to Zn have been shown to accu-mulate this metal (Burken and Schnoor, 1996).

5. Factors affecting heavy metal uptake by plants

The uptake of heavy metals by plants depends on several factorssome of which are discussed below:

5.1. Soil factors

Several edaphic factors like sorptive capacity of soil, heavymetalcontent, cation exchange capacity, soil pH and organic mattercontent affect metal hyperaccumulation in plants (Alloway, 1995;Tiller et al., 1995; Cheng, 2003; Chaney et al., 2007). Generally, onlya fraction of soil metal is readily available (bioavailable) for plantuptake since the bulk of soil metals are commonly found as insol-uble compounds unavailable for transport into roots (Lasat, 2002).Cations of heavy metals are often bound to soil particles because ofsoil cation exchange capacity. The cation exchange capacity isa measure of the soil’s capacity to exchange ions. The negativecharges are supplied by clay and organic matter of the soil. Thebinding affinity of cations reduces cation movement in vascularplants. Thus, the higher the cation exchange capacity (CEC) of thesoil, the greater the sorption and immobilization of the metals.

Metal solubility and availability are dependent on soil charac-teristics and are strongly influenced by soil pH, which is consideredas the major factor influencing the availability of elements in thesoil for plant uptake. Plants absorb mineral elements in the ionicform in solution, and the presence of these forms is stronglyinfluenced by matrix pH (Dzantor and Beauchamp, 2002). A lowersoil pH increases concentration of heavy metals in the solution bydecreasing their adsorption. In soil, the solution concentrations ofmetal contaminants tend to increasewith decreasing pH because oftheir displacement from exchangeable sites on solid surfaces byincreasing activity of hydrogen ions as there is a decrease in pH.This can increase the availability of the contaminant for plantuptake, but can also result in concentrations of elements at levelsthat are toxic to the plant. Many metal cations like Cd, Cu, Hg, Pb,and Zn are reported to be more soluble and available in the soilsolution at low pH (below 5.5) (Blaylock and Huang, 2000). Theincreased availability of metals at low pH has led phytoextractionresearchers to study incorporation of acidifiers (NH4 containingfertilizers, organic and inorganic acids, and elemental S) into metalcontaminated soils to improve the success of phytoextraction.However, inspite of the promise of some acidifying agents, littleresearch has been carried out on this subject and this needs furtherinvestigation.

The organic content of the soil also has a strong bearing on theextent of phytoextraction of heavy metals. The addition of peat andmanure is reported to increase Cu, Zn and Ni accumulation inwheat(Narwal and Singh, 1998). Peat and manure are quite heteroge-neous substances that can concurrently exert mobilizing andstabilizing effects (Schmidt, 2003). Acid peat reduces soil pH, whichincreases concentration of soluble metals in the soil. It alsoincreases the CEC of soils, provides sorption sites, reduces metalmobility and promotes higher binding affinity (Schmidt, 2003).

The oxidation state of a metal contaminant also determines itssolubility and relative availability for uptake by plant systems. In

Table 3Expression of metallothionein genes in transgenic plants.

MT gene Source Plant species genetically modified Reference

mt-IA Mouse (Mus musculus) Nicotiana tabacum Pan et al. (1994)mt-b-glucuronidase fusion Chinese hamster (Cricetulus griseus) Nicotiana tabacum Hattori et al. (1994)mt-II Humans (Homo sapiens) Nicotiana tabacum de Borne et al. (1998)cup1 Yeast (Saccharomyces cerevisiae) Nicotiana tabacum Thomas et al. (2003)tymt Cattail (Typha latifolia) Arabidopsis thaliana Zhang et al. (2004)hmt Humans (Homo sapiens) Medicago varia Watrud et al. (2006)mt-1 Mouse (Mus musculus) Lycopersicon exculentum Sheng et al. (2007)smtA Cyanobacteria (Synechococcus sp.) Arabidopsis thaliana Xu et al. (2010)femt3 Buckwheat (Fagopyrum sp.) Nicotiana debneyii Nikolic et al. (2010)ccmt1 Pigeon pea (Cajanus cajan) Arabidopsis thaliana Sekhar et al. (2011)psmtA Pea (Pisum sativum) Populus alba Turchi et al. (2012)


general, the oxidized forms of most common metal contaminantsare less soluble and consequently less available for plant uptake,chromium being the exception.

5.2. Other environmental factors

The climate at a particular site poses the greatest and mostobvious limitation to the applicability of phytoextraction. Temper-ature influences transpiration, water chemistry, growth andmetabolism of plants and therefore, both uptake and elimination ofpollutants (Yu et al., 2005). The removal rates of metals by plantsare known to increase linearly with increase of temperature(Yu et al., 2010). Baghour et al. (2001) found that potato plants(Solanum tuberosum L.) showed higher uptake of Cr at hightemperature as compared to plants grown at low temperature. Yuet al. (2010) investigated the removal of chromium from hydro-ponic solution by hybrid willows plants to changes in temperatureranging from 11 to 32 #C and found the highest removal rate at32 #C. Temperature dependency of phytoremediation has been oneof the major limitations of the technology, especially in temperateregions where most field bioremediation is limited to the warmerparts of the year. Technologies currently exist for warming upcontaminated matrices to allow bioremediation implementationduring cold periods, but it is questionable whether adoption of suchenergy-demanding approaches can still make bioremediation thefavoured, cost effective alternative to other remediation schemes(Dzantor and Beauchamp, 2002).

Environmental pollution may also exert complex influence onmineral accumulation as the plant roots absorb heavy metals fromthe soils and aerosols penetrate from the atmosphere into theplants through the surface of leaves. Minute metal particles stick tothe leaves but only some are absorbed. For example, Pb remains assurface precipitate, while Cu, Cd and Zn can partially penetrate intothe leaves (Kabata-Pendias and Pendias, 1992).

5.3. Genetic factors

Plant genotype is considered as the most important factoraffecting heavy metal uptake by plants. A range of plant familiesdistributed over areas diverse geographically, but possessinga common characteristic of natural enrichment for some specificmetal is known (Prasad and Freitas, 2003). At the species level,numerous reports have confirmed the existence of significantgenotypic differences in the heavy metal uptake and distributionbetween and within species, and even within cultivars (Chardotet al., 2005; Liu et al., 2005; Grant et al., 2007; Bhargava et al.,2008a; Richau and Schat, 2009; Kramer, 2010; Hanikenne andNouet, 2011). Some genotypes respond positively to increasedheavy metal concentration in soil, while others may be inert orshow negative growth. In some cases, both the genomes of anamphidiploids acting together are responsible for high metalaccumulation, like the AABB genome in B. juncea (Nanda-Kumaret al., 1995).

The existence of metal accumulators demonstrates that someplants have the genetic potential to clean contaminated soils. It hasbeen demonstrated that tolerance to high levels of metals byhyperaccumulators is under genetic control (Macnair, 1993), whichallows the plants to produce specific molecules that react withmetals to form complexes. These complexes can then be storedaway from sensitive tissues. Understanding the genetic mechanismof metal accumulation in hyperaccumulator species is importantbecause it facilitates the use of various approaches to geneticimprovement of plants for metal uptake. The genetic control ofheavy metal accumulation in plants is not well understood. Earlierworks suggested thatmetal tolerance is complex and is the result of

the action of a large number of genes (Antonovics et al., 1971).However, recent studies have shown that metal tolerance is regu-lated by a few major genes or sometimes by a single gene (Macnair,1993; Clarke, 1995; Macnair et al., 2000). The hybridization of Zntolerant and non-tolerant species has brought out the fact that bothtraits are genetically independent, and Zn tolerance is controlled bya single gene. Several researchers have conducted interestinggenetic studies with the species A. halleri that is considered close toArabidopsis thaliana and has undergone a natural selection for Zntolerance (Bert et al., 2000; Roosens et al., 2008a, 2008b). A. halleriis a Zn hyperaccumulator that is small in size and hyper-accumulates Zn from Zn rich soils (Table 2). Zn tolerance andhyperaccumulation have been found to be constitutive in A. halleri(Chaney et al., 2007). Macnair et al. (1999) made crosses betweenA. halleri and Arabidopsis lyrata subsp. petraea and subsequentbackcrosses to allow evaluation of the inheritance. From the anal-ysis of the F2 generation it was found that Zn hyperaccumulationand tolerance were separate genetic properties under independentgenetic control in A. halleri. Bert et al. (2003) analyzed the co-segregation of Cd tolerance and Cd accumulation in the progenyfrom an A. halleri $ A. lyrata subsp. petraea F1 plant backcrossed toA. lyrata subsp. petraea (BC1). It was found that these charactersinherited independently, but the presence of co-segregation of Cdtolerance with Zn tolerance, and Cd accumulation with Zn accu-mulation suggested partial pleiotropic control of these characters.Thus, Zn tolerance appeared to be controlled by a single gene, whileCd and Zn hyperaccumulation was controlled by several genes(Macnair et al., 1999; Bert et al., 2003). Evaluation of differencesbetween A. halleri accessions has shown small variation in metalaccumulation, but no apparent difference in inheritance of metalaccumulation (Bert et al., 2000).

Isolation of the quantitative trait loci (QTL) associated withmetal tolerance holds great promise for the identification of thegenes responsible for this adaptation (Roosens et al., 2008a, 2008b).Mapping of quantitative trait loci (QTLs) for Zn and Cd tolerance hasshown 3 additive QTLs for Zn and Cd tolerance, with trait-enhancing alleles originating from the A. halleri parent (Courbotet al., 2007; Willems et al., 2007). The co-location of 1 QTL for Cdtolerance with a Zn tolerance locus has confirmed the hypothesis ofpartial pleiotropic control of Zn and Cd tolerance (Bert et al., 2003).Deniau et al. (2006) mapped QTLs for Cd and Zn accumulation in anF2 cross between 2 plants and found 1 common locus for Cd and Znin root, 1 for Cd in root and Cd in shoot, and 1 for Zn in root and Znin shoot. The trait-enhancing alleles at the 3 Cd accumulation lociwere all derived from the parent having greater Cd accumulationcapacity, while both parents contributed trait-enhancing alleles tothe Zn accumulation loci. Deniau et al. (2006) found transgressivesegregation for Zn accumulation, but not for Cd accumulation. Theaccumulation rates of Zn and Cd in the F2 plants were significantlyphenotypically correlated and governed by common geneticdeterminants, as well as additionally by more metal-specificdeterminants. Segregation studies in F3 and F4 families derivedfrom a cross between T. caerulescens accessions have shown that Znand Ni accumulation are pleiotropically controlled by the samegenes (Richau and Schat, 2009). The high-throughput technologieslike microarray support the idea that genes that are thought to beinvolved in hyperaccumulation and hypertolerance are not species-specific or novel, but rather differently expressed and regulated,compared with non-hyperaccumulator species (Verbruggen et al.,2009).

6. Plant improvement for enhanced phytoextraction

The goal of remediating metal contaminated soil is generally toextract the metal from the large soil volume and transfer it to


a smaller volume of plant tissue for harvest and disposal. This isdue to the fact that metals cannot be metabolized or broken downto less toxic forms. The amount of pollutant a plant can removefrom the soil is a function of its tissue concentration multiplied bythe quantity of biomass formed (Macek et al., 2007). Low yield andslow growth rates have been cited as limiting factors for thedevelopment of effective metal phytoremediator plants (Brownet al., 1995). Most of the known metal accumulating plants aremetal selective, show slow growth rate, produce relatively littlebiomass and can be used for phytoextraction in their naturalhabitats only (Kamnev and van der Lelie, 2000). Thus, while theamounts of metal concentration per unit of plant biomass can behigh, the total amounts of metal removed at a site during a givenperiod can be quite low. For example, although T. caerulescens cantake up sufficient levels of metals to make harvesting and metalrecovery economical, they are often limited by their small biomass(Meagher et al., 2000; Nedelkoska and Doran, 2000). Moreover,the use of hyperaccumulator plants can be limited because ofless information about their agronomic characteristics, pestmanagement, breeding potential and physiological processes(Cunningham et al., 1995). However, a rapidly growing non-accumulator could be modified to enable it to achieve someof the properties of hyperaccumulators. Two approaches arecurrently being explored to develop and/or improve the metalaccumulating plants:

1) Conventional breeding, and 2) genetic engineering.

6.1. Conventional breeding

Conventional breeding approaches coupled with suitableagronomic practices like soil fertilization and conditioning, properplant density, crop rotation, weed control and irrigation practicescan go a long way in enhancing the phytoextraction capacity vis-à-vis metals (Lasat, 2000). Traditional plant breeding uses theavailable genetic diversity within a species to combine the traitsneeded for successful phytoextraction (Li et al., 2003). Althoughthere have been efforts to study genotypic differences in metaluptake and biomass in hyperaccumulator species, little effort hasbeen made to breed these species for domestication. Efficientmanagement and utilization of germplasm requires detailedknowledge of the genetic diversity of agronomic traits for propercharacterization of populations to facilitate efficient synthesis ofbreeding populations that are designed to accomplish specificobjectives (Bhargava et al., 2007, 2008a). In any crop-breedingprogram, assessment of genetic diversity is invaluable as it helpsin the identification of diverse parental combinations to createsegregating progenies with maximum genetic variability andfacilitates introgression of desirable genes from diverse germ-plasm into the available genetic base (Bhargava et al., 2007). Theexisting genetic diversity in crops can be used for phytoextractionby identifying easily cultivable, high biomass yielding plants andpracticing selection in future generations. Numerous reports areavailable which suggest the manipulation of germplasm and useof selection strategies to reduce heavy metal concentration indifferent plant species (Li et al., 1997; Liu et al., 2005; Bhargavaet al., 2008a). Apart from selection strategy, conventionalbreeding methodology also involves the transfer of hyper-accumulator phenotype like metal tolerance and increased uptakefrom small, slow growing, hyperaccumulator species to fastgrowing, high biomass producing non-accumulator plantsthrough hybridization (Chaney et al., 2000; Grant et al., 2007;Bhargava et al., 2008a, 2008b).

Researchers all over the world are looking for new plant specieshaving natural variability for heavy metal accumulation. Recently,there have been reports on the utilization of the available genetic

diversity in underutilized crops for phytoextraction (Lasat et al.,1998; Tamura et al., 2005; Bhargava et al., 2008a). Commonbuckwheat (Fagopyrum esculentum Moench) is the first known Pbhyperaccumulator with high biomass productivity (Tamura et al.,2005). F. esculentum is reported to naturally accumulate up to4200 mg/g of Pb in the shoot. Amending the soil with methyl-glycinediacetate (MGDA), a biodegradable chelator, resulted ina 5-fold increase in the Pb shoot concentration. These findingsqualify this species as an excellent candidate for remediatingPb-contaminated soils. The red root pigweed (Amaranthus retro-flexus L.) is known to accumulate soil 137Cs to levels which maysupport a phytoextraction technology, while other crop plants areless able to accumulate Cs (Lasat et al., 1998). Studies conducted onanother underutilized pseudocereal, Chenopodium, indicateda large genetic variation for various heavy metals at species andsub-species levels, coupled with high biomass (Bhargava et al.,2008a). At the species level, Chenopodium quinoa showed highaccumulation of Zn, Cr and Cd, while Chenopodium album and theAmerican species Chenopodium bushianum accumulated highamounts of Ni (Bhargava et al., 2008a). This genetic diversity couldbe effectively exploited in breeding crops with high phytoex-traction properties.

Somaclonal variation offers the possibility to obtain changes inone or a few characters of an otherwise outstanding cultivarwithout altering the remaining and often unique part of thegenotype. One future line of action can be to utilize somaclonalvariation for development of plants having improved phytoex-traction capacity. Symmetric and asymmetric somatic hybridiza-tions have recently gained importance as genetic modifiers.Brewer et al. (1999) raised somatic hybrids (protoplast electro-fusion) between Zn hyperaccumulator T. caerulescens and Brassicanapus and found that the hybrids accumulated high levels of Znthat would otherwise have been toxic to B. napus. Some of thehybrids produced high biomass combined with high metal accu-mulation and tolerance, making them attractive for Zn phytoex-traction. This study clearly indicates that transfer of metalhyperaccumulating phenotype is feasible. Likewise, Gleba et al.(1999) also obtained somatic hybrids from a cross betweenB. juncea and T. caerulescens that had significant Pb accumulatingproperties. Metal resistant traits were introduced into the high-biomass Pb accumulator B. juncea using somatic hybridization.T. caerulescens, a known Ni and Zn hyperaccumulator, was selectedas one of the parents for both symmetric and asymmetric hybridsin which T. caerulescens protoplasts were irradiated before fusion.Eighteen hybrids were regenerated of which 2 were found to befertile. One of these hybrids had vigorous growth, characteristic ofB. juncea and increased resistance when grown in Pb, Ni, and Zncontaminated soil. The total amount of Pb phytoextracted by eachhybrid plant was much greater because of the large biomassproduced on the contaminated soil. Several other researches havealso reported the utilization of somatic hybridization to introducetoxic metal resistant traits present in T. caerulescens into B. juncea(Dushenkov et al., 2002; Alkorta et al., 2004). The hybrids soobtained have demonstrated high metal accumulation potential,tolerance to toxic metals, and good biomass production. In vitrobreeding and somaclonal variation have been used to improve thepotential of Indian mustard (B. juncea) to extract and accumulatetoxic metals (Nehnevajova et al., 2007). Calli from B. juncea werecultivated on a modified MS medium and somaclones wereregenerated from metal-tolerant callus cells. Seven out of thirtyindividual variants showed higher metal extraction than thecontrol plants. The improvement of metal shoot accumulation ofthe best regenerant and metal extraction indicated that B. junceacould be used for phytoremediation purpose (Nehnevajova et al.,2007).


6.2. Genetic engineering

Several anatomical constraints severely restrict sexual compat-ibility between taxa and pose serious limitations in developinghybrids with increased phytoextraction capability. Biotechnologyhas opened new gateways in phytoremediation technology byoffering the opportunity for direct gene transfer and overcomingthe limitations imposed by sexual incompatibility. The develop-ment of transgenic plants with increased metal uptake, accumu-lation and tolerance to toxicity is nowbeing considered a promisingalternative (Fig. 1). Genetic engineering is a technique that can beapplied advantageously to the development for ideal phytor-emediation plants that combine high metal accumulating capacityand high aboveground biomass yield (Kärenlampi et al., 2000).With the use of genetic engineering, it is feasible to manipulatea plant’s capacity to tolerate, accumulate, and/or metabolizepollutants, and to create an ideal plant for environmental cleanup.Recent progress in determining the molecular basis of metalaccumulation and tolerance by hyperaccumulators has provided uswith a strong scientific basis to outline some strategies forachieving this goal. Many genes are reported to be involved inmetal uptake, translocation, sequestration, chemical modification,and tolerance (Table 4). The introduction and overexpression of thehyperaccumulating genes into a non-hyperaccumulator plant couldbe a possibleway to enhancemetal uptake, accumulation, toleranceand detoxification process (Clemens et al., 2002). The over-expression of genes encoding the rate-limiting gene product isexpected to lead a faster overall rate of the pathway and to moreefficient phytoremediation (Pilon-Smits and Pilon, 2002). Besidesthis, the repression of an endogenous gene by inserting a gene ofreverse orientation (antisense technology) can also result inenhanced metal uptake by plants (Shah and Nongkynrih, 2007).The introduction of an additional metal binding domain to theimplemented protein further enhances the metal binding capacity(Macek et al., 1996; Kotrba et al., 1999).

The fact that plants can benefit from organic acid exudation ina number of ways has aroused interest of biotechnologists toincrease organic acid exudation in crop and pasture species (Ryan

et al., 2001). Several reports on transgenic plants tolerant to thepresence of toxic levels of metals have appeared in recentyears (Table 5). In most of the studies, the overexpression of thegenes encoding for the enzymes of phytochelatin synthase, ACCdeaminase, S-metabolism, glutathione, Hg2þ-reductase, arsenatereductase, aldolase/aldehyde reductase, enzymes of histidinebiosynthesis and metallothionein (MT)-genes have been effectivelycarried out (Shah and Nongkynrih, 2007). The bioengineering oftransporter genes to manipulate the transport of metal ions insidethe cell has also been successfully exploited and a combination ofthese genes in rapidly growing plant species has led to promisingresults.

Several strategies have been successfully used to create trans-genics that show promising properties for phytoremediation.Table 5 provides an overview of the different approaches involvedin the production of transgenics for enhancing phytoextractioncapacity of crop plants for different heavy metals. One strategy forincreasing the efficiency of phytoextraction involves increase in themetal translocation to the shoot by increasing plant transpiration.Gleba et al. (1998) observed that the genetically modified plants ofB. juncea with an increased transpiration phytoextract 104% morelead than the wild type plants, making it a good candidate for fieldoptimization and use. A simple and direct method for enhancingthe effectiveness of phytoextraction is to overexpress in transgenicplants, the genes involved in metabolism, uptake, or transport ofspecific pollutants. The introduction of these genes has beensuccessfully achieved using Agrobacterium tumefaciens-mediated

Search for metal accumulators/organisms living in high metal stress

Isolation and cloning of genes conferring metal tolerance and/or accumulation

Introduction of gene in the target plant; Field trials

Study organism at low and high metal

stress

Comparison with close relatives

Characterization of gene products in a

suitable model system

Introduction of the gene in a model plant; Stress

induced studies

Transgenic plant for phytoremediation

Fig. 1. Development of metal tolerant/accumulator plant using genetic engineering (Adapted from Kärenlampi et al., 2000).

Table 4Some important genes involved in metal tolerance and accumulation.

Metal Gene Reference

Al aha2 Moffat (1999)Fe frd3, Durrett et al. (2007)Zn mtp1, mtp3 Gustin et al. (2009)Cu cbf Szira et al. (2008)Cd atcax2, atcax 4, mt1 Pan et al. (1994); Korenkov et al. (2007)Ni reg2, sat-c Schaaf et al. (2006); Na and Salt (2011)


plant transformation. The overexpression of glutamylcysteinesynthetase has been accomplished by genetic engineering in Pop-ulus angustifolia, Nicotiana tabacum and Silene cucubalus whichenhances heavy metal accumulation as compared to the wild typeplants (Fulekar et al., 2009). Tomato plants have been geneticallymodified to express 1-amino-cyclopropane-1-carboxylic acid (ACC)deaminase, a bacterial gene which resulted in enhanced metaltolerance and increased uptake of a range of heavy metals (Grichkoet al., 2000). The overexpression of Escherichia coli gshI geneencoding g-glutamylcysteine synthetase in B. juncea resulted inincreased concentration of Cd (up to 90%) in transgenic plants ascompared to wild types (Zhu et al., 1999). Another promisingapproach is to overexpress enzymes catalyzing rate-limiting steps.ATP sulfurylase (APS) is a rate-limiting enzyme in the seleniumdetoxification process which facilitates the reduction of selenate toselenite. The overexpression of an ATP sulfurylase (APS) fromA. thaliana in B. juncea led to three times more uptake and accu-mulation of metals in the transgenic plants as compared to the wildtypes (Pilon-Smits et al., 1999). In another study, Indian mustardplants overexpressing cystathionine gamma synthase (CGS) weredeveloped. It was observed that the transgenics had enhancedtolerance to selenite and volatilized Se two to three times fasterthanwild type, while at the same time accumulating less Se in rootsand shoots (van Huysen et al., 2003). However, inspite of the initialsuccess with B. juncea, the plant has never been shown to be usefulin field phytoextraction. It has poor yield and rapidly flowers ifgrown under warm conditions. Also the plant has no metal toler-ance and no metal accumulation in absence of added chelators.

Modification or overexpression of the enzymes that are involvedin the synthesis of PCs is a promising approach to enhance heavymetal tolerance and accumulation in plants and has been tried togenetically transform high biomass plants into efficient phytor-emediators (Zhu et al., 1999). Several attempts have been made toincrease the formation of PCs by overexpressing genes encodingenzymes that could stimulate the synthesis of cysteine and gluta-thione. The induction of PCs by Cd suggests that increasing

biosynthesis of phytochelatins would improve tolerance and phy-toextraction, but there has been no supporting evidence that thisapproach would yield a Cd phytoextraction plant. Cd tolerance hasbeen increased to a maximum 3e7 fold by high expression of PCsynthase (Heiss et al., 2003), which is trivial as compared to the 200times higher tolerance of Zn and Cd of T. caerulescens (Chaney et al.,2005; Wang et al., 2006). Similar findings have been observedwhen researchers tried to alter Cd accumulation in tobacco shootsby transgenic expression of phytochelatin or metallothioneins,despite extensive testing of many constructs (Lugon-Moulin et al.,2004).

For some metals such as Hg and selenium, a promising strategyis to convert the metal to a volatile form for release and dilutioninto the atmosphere. The most spectacular application of biotech-nology in phytoremediation has been the engineering of plantscapable of removing methyl-Hg from contaminated soils (Rughet al., 1996; Pilon-Smits and Pilon, 2000). Arabidopsis plants werethe first to be engineered with a gene, mercuric ion reductase(merA), from a bacterium that was resistant to Hg (Rugh et al.,1996). Later, transgenic Arabidopsis plants were raised whereinmerB enzyme was targeted to the endoplasmic reticulum(Bizily et al., 2003), which showed a 10e70 times higher specificactivity to degrade organic Hg than transgenic plants withcytoplasmic merB. This was followed by transformation of yellowpoplar (Liriodendron tulipifera) and Eastern cottonwood (Populusdeltoides) with merA, leading to increased tolerance to ionic Hg(Rugh et al., 1998; Che et al., 2003). Transgenic cottonwoodshoots had normal growth on medium containing 25 mM Hg (II),a concentration of Hg that killed the wild-type shoots. The trans-genic plants produced up to 4-fold more elemental Hg than wild-type plants, demonstrating that the plants take up and transformHg to the less toxic form. Successful transformation of rice (Oryzasativa) has been achieved for enhanced Hg remediation (Heatonet al., 2003). The merA transgenic rice tolerated concentrations ofHg2þ that killed the wild-type controls and steadily converted theHg2þ to its less toxic volatile form (Heaton et al., 2003).

Table 5Transgenic approaches for enhancement of heavy metal uptake in different plants.

Metal Plant Methodology/Approach Reference

Ni Brassica napus Arabidopsis Expression of bacterial ACC deaminase gene Stearns et al. (2005)Expression of nicotianamine synthase cDNA (TcNAS1) Pianelli et al. (2005)

Cd Brassica juncea Overexpression of gamma-glutamylcysteine synthetase and glutathione synthetase Bennett et al. (2003)Reisinger et al. (2008)

Arabidopsis Overexpression of Arabidopsis phytochelatin synthase (AtPCS1) Lee et al. (2003)Arabidopsis Expression of yeast ABC transporter family member, YCF1 Song et al. (2003)

Pb Arabidopsis Expression of yeast ABC transporter family member, YCF1 Song et al. (2003)Brassica juncea Overexpression of a yeast cadmium factor 1 (YCF1) Bhuiyan et al. (2011a)Brassica juncea Overexpression of ATP-binding cassette (ABC) transporter gene AtATM3 Bhuiyan et al. (2011b)

Cu Nicotiana tabacum Expression of yeast metallothionein (CUP1) gene Thomas et al. (2003)Populus alba Expression of PSMTA1 gene from Pisum sativum Balestrazzi et al. (2009)Arabidopsis Expression of yeast copper-dependent transcription factor ACE1 Xu et al. (2009)

Zn Arabidopsis Overexpression of Zn Induced Facilitator 1 (ZIF 1) Haydon and Cobbett (2007)Brassica juncea Overproduction of the g-glutamylcysteine synthetase and glutathione synthetase Bennett et al. (2003)Nicotiana tabacum Overexpression of glyoxalase pathway enzymes Singla-Pareek et al. (2006)

Hg Arabidopsis Expression of bacterial gamma-glutamylcysteine synthetase gene under controlof a strong constitutive actin regulatory sequence (A2)

Li et al. (2005)

Nicotiana tabacum Expression of ppk gene specified bacterial polyphosphate (polyP) Nagata et al. (2006)Nicotiana tabacum Expression of a modified bacterial Hg reductase, merA gene Heaton et al. (2005)

As Arabidopsis Expression of bacterial gamma-glutamylcysteine synthetase gene under controlof a strong constitutive actin regulatory sequence (A2)

Li et al. (2005)

Brassica juncea Overexpression of ATP sulfurylase Wangeline et al. (2004)Brassica napus Expression of Enterobacter cloacae UW4 1-aminocyclopropane-1-carboxylate (ACC)

deaminase (EC 4.1.99.4) geneNie et al. (2002)


Bizily et al. (2000) accomplished the full pathway from methylHg to the least toxic metallic Hg by expressing both merA andorganomercurial lyase (merB) within the same plant. Arabidopsisplants transformed with both genes were tolerant to concentra-tions of methyl-Hg 50 times higher than the concentrations towhich wild-type plants were tolerant and 10 times higher thanthose to which plants transformed with merB alone were tolerant.Transformation of Spartina alterniflora, the common wetlandplant, with both merA and merB has been achieved, resulting inincreased tolerance of the transgenic plants to phenylmercuricacetate and mercuric chloride (Czako et al., 2006). Recently,Eastern cottonwood was transformed with both merA and merB(Lyyra et al., 2007). These transgenic cottonwoods were stronglyresistant to toxic phenylmercuric acetate, and had an increasedrate of detoxification that was 2e3 times faster than that of thecontrol plants. In the transgenics,merB catalyzes the protonlysis ofthe CeHg bond with the release of Hg2þ, which is subsequentlyconverted by merA to Hg0, a less toxic volatile element that isreleased into the atmosphere. Such genetically modified plantsrelease 10 times of elemental Hg as compared to non-transformedplants.

Following the transgenic approach, only Hg has been demon-strated to work in the field (Heaton et al., 2003), but publicacceptance has been difficult because Hg0 is volatilized at the soilsurface and will eventually be re-deposited on soil or water(Chaney et al., 2007). Unfortunately, the contaminant was notdestroyed but merely transformed from soil-bound ionic Hg2þ toairborne elemental Hg0.

Clonal propagation has opened the door to the creation of tree‘remediation’ cultivars i.e. perennial trees having excellent metaluptake (Stomp et al., 1993).Work is underway to screen tree speciesfor their ability to tolerate, take up, translocate, sequester, anddetoxify heavy metal ions. Clone stability and in vitro phytoex-traction capacity of vegetative clones of Populus $ canescens thatincluded two transgenic clones (ggs11 and lgl6) were studied asin vitro leaf disc cultures (Gyulai et al., 2005). The transgenic poplarcyt-ECS (ggs11) clone, as stimulated by the presence of Zn, showedelevated heavy metal (Cu) uptake as compared to the non-transformed clone. This suggests that gshI-transgenic poplarsmay be suitable for phytoremediation of soils contaminated withZn and Cu (Gyulai et al., 2005). Future research may focus onoptimizing metal accumulation in cloned plants and developingclones of trees that exhibit a high degree of accumulation and largerbiomass.

7. Limitations

Phytoextraction seems to hold promise for the remediation ofmetal polluted soils. Nevertheless, ongoing research reveals thatthe applicability of the technique is debatable since the practicalimplications are not so evident (van Nevel et al., 2007). Presently,the technology is limited due to long period required for cleanup,restricted number of target metals that can be extracted, limiteddepth that can be assessed by the roots, decline in phytoextractionefficiency under increasing metal concentrations and the lack ofknowledge on the agronomic practices and management (Kelleret al., 2003; Ernst, 2005; McGrath et al., 2006; Robinson et al.,2006; Audet and Charest, 2007). Also, the complexity of hyper-accumulation has not been fully understood, either at the tissue orat the subcellular level. Phytoextraction seems possible for only Asand Ni, while for the other metals the technology still appears to befar from practice. The vast majority of the hyperaccumulatorspecies discovered so far are Ni hyperaccumulators (Table 1), whilespecies accumulating Cu, Pb, Cd, Zn, Co and As are much lessnumerous (do Nascimento and Xing, 2006).

Most of the data on the performance of phytoremediatingtransgenic plants are based on observations made in controlledconditions (laboratory) often on growth media, rather than in thefield. Therefore, it is important to confirm the performance ofphytoremediation systems on large-scale contaminated sites. Thebioavailability of the contaminants on the contaminated sitesappears to be a major factor in the discrepancy between lab andfield conditions. A better understanding of soil properties and thephysio-chemical factors influencing the solubility of toxiccompounds is likely to help in the improvement of on-site plantperformances in the future (Singh et al., 2003). There is also anurgent need to gain indepth knowledge about the molecularmechanisms that allow plants to remediate polluted soils, partic-ularly with respect to hyperaccumulation and hypertolerance. Theidentification of genes involved in the acquisition and the homeo-stasis of toxic materials, along with an understanding of the waythey are regulated is likely to improve phytoremediation tech-nology to a great extent (Cluis, 2004).

8. Is phytoextraction commercially viable?

Metal-contaminated soil can be remediated by physical, chem-ical or biological techniques. Chemical and physical treatments ofmetal contaminated soils irreversibly affect soil properties, destroybiodiversity and may render the soil useless as a medium for plantgrowth (Padmavathiamma and Li, 2007). There is a need to developsuitable cost-effective biological soil remediation techniques toremove contaminants without affecting soil fertility. Phytoex-traction is one of the low cost techniques for contaminated soilremediation (Table 6).

In the absence of long-term site trials, phytoextraction efficiencyshould be evaluated using models. Phytoextraction projects can beevaluated on the amount of metals being removed from the soil inrelation to the total amount present in the soil (Mertens et al., 2005,2006) and on the time needed for remediation. However, assess-ment of feasibility of phytoextraction based on modelling has itsown limitations. It has been suggested that the biomass productionmight decrease over time due to nutrient depletion in the soil afterseveral croppings or pest infections (van Nevel et al., 2007). Tomitigate this, fertilizers, pest control and crop rotation might benecessary. In natural conditions, plant growth may be limited byother environmental variables, such as low pH, low water avail-ability, salinity or insufficient aeration, whereas experimentalconditions are generally optimal. Lastly, a large area of metal-contaminated soils is polluted with more than one element,while few plant species can extract high concentrations of morethan one element (Ernst, 2005). This polymetallicity stronglyaffects the productivity even of metal resistant plants, causing theextraction period to become too long to be economically feasible(Ernst, 2005; Robinson et al., 2006).

Development of a commercially viable technology for usinghyperaccumulator plant species to phytoextract metals requires theidentification or creation of an ideal phytoextraction plant, opti-mization of soil and crop management practices, and development

Table 6Cost comparison of different cleanup techniques for metalpollution (Glass, 1999b).

Process Cost (US $/t)

Land filling 100e500Vitrification 75e425Chemical treatment 100e500Electrokinetics 20e200Phytoextraction 5e40


of methods for biomass processing and extraction (Li et al., 2003).Phytoextraction can be applied either to metal-contaminated soilsor to ores that cannot be economically extracted by traditionalmining technology. In the former case, phytoextraction is a type ofphytoremediation, while the term ‘phytomining’ has been appliedto the latter case where the economic value of the recovered metalis the primary motive (Li et al., 2003). The value of the phytoex-tracted metal, if it is recycled, can help to defray the costs of phy-toremediation. Phytomining includes the generation of revenue byextracting soluble metals produced by the plant biomass ash, alsoknown as bio-ore. If phytoextraction could be combined withbiomass generation and is commercially utilized as an energysource, then it could be turned into a profitable operation, with theresidual ash available to be used as an ore (Cunningham and Ow,1996; Brooks, 1998; Padmavathiamma and Li, 2007). With somemetals like Ni, Zn and Cu, the value of reclaimed metal may providean additional incentive for phytoremediation (Chaney et al., 1997;Thangavel and Subhuram, 2004). The total world remediationmarket is reported to be approximately US $15e18 billion per year(Memon and Schroder, 2009). Several companies and researchgroups around the world are pursuing phytomining strategies.B. juncea and other plant species are reported to accumulate asmuch as 20 mg/kg of gold in greenhouse experiments when plantswere supplemented with a solubilizing agent (ammonium thiocy-anate) (Prasad, 2003). Some commercial companies are gainingeconomic benefits from phytoextraction not just by recoveringextracted metals from plant biomass, but also by using the biomassfor energy generation (Glass, 1999a, 2000). There is evidence forextraction of high purity Ni fromNi-contaminated Alyssum biomass(Bani et al., 2007; Chaney et al., 2008). The commercially viableoptions include either the recycling of Ni rich biomass in metal-lurgy or use as Ni fertiliser to correct Ni deficiency in field crops. Asignificant number of companies based on phytoextraction haveemerged that have created growing industries in North Americaand in several European countries. Smaller, but emerging marketsexist in developing nations, particularly in portions of Asia. Thereare about 10 companies in Canada and 20 in Europe which areconducting research or which have carried out commercial reme-diation using phytoremediation or related technologies. Numerouscommercial projects have been undertaken in several countriesaround the world though some of these companies are pursuingphytoremediation at the research stage only (Glass, 2000).

9. Prospects

The advantages of using metal accumulating plants for theremoval of metals from contaminated soils include lower costs,generation of recyclable metal-rich plant residue, applicability toa range of toxic metals, minimal environmental disturbance andpublic acceptance. Plants with increased metal accumulationproperties may also be utilized to enhance crop productivity inareas with suboptimal metal levels, or as fortified food and feed(Guerinot and Salt, 2001). But, inspite of these advantages, phy-toextraction is still an emerging technology and concerted effortsare needed if this environmental friendly technology is to beexploited.

The sequencing of complete genome of hyperaccumulatorscould go a long way in identifying promising functional noncodingregions and to narrow the focus for experimental tests (Wray andBabbitt, 2008). The discovery of metal related genes with the aidof genome sequencing will open up new avenues for the creation oftransgenics having desired properties that would help in estab-lishing phytoextraction as a potent technology for environmentalcleanup. The understanding of genome evolution in the hyper-accumulators should be improved by merging ecological and

molecular genomics (Verbruggen et al., 2009). The search forsignatures of recent adaptive evolution across candidate genes formetal tolerance or accumulation could be a promising approach.Apart from constitutive overexpression of a single gene, severalgenes may be expressed simultaneously in specific cellularcomponents under specific conditions. Several approaches could befollowed to achieve this goal. These include identification of themetal transporter proteins and introducing genes encoding trans-porter molecules to enhance the ability of metal ions to enter plantcells (Tong et al., 2004); overexpression of the enzyme phytoche-latin synthase (PS) in plants that would lead to increased metaltolerance and accumulation; overproduction of nicotianamine tomanipulatemetal translocation and tolerance as well as iron uptakein cereals (Pilon-Smits and Pilon, 2002); overproduction of histi-dine (His) since the genes involved in His biosynthesis have beencloned (Persans et al., 1999) and preliminary studies suggestingthat histidine overproducing plants have enhanced Ni tolerance(Kramer and Chardonnens, 2001).

However, the use of transgenics in nature for phytoextractionshould be preceded by a thorough risk assessment study on a case-by-case basis and weighing the benefits and risks as compared toalternative technologies. For phytoremediation to be a viable, costeffective alternative, plants will need to alter or detoxify thecontaminants rather than simply accumulate or displace them.Theoretical assessment of risks associated with the use of metal-volatilizating plants has pointed that the use of transgenic plantshaving phytoextraction capacity is relatively safe (Lin et al., 2000;Meagher et al., 2000; Rugh et al., 2000). Still, concerns have beenraised about the safety of phytoextraction that range from metalsentering the food chain through herbivores, accumulation of metalsin the topsoil and dispersion of plant material to adjacent envi-ronments (Perronnet et al., 2000; Linacre et al., 2003; Mertenset al., 2005, 2007). Although the risk of transgenic plants or theirgenes escaping into the environment is not considered to bea significant problem, the transgenic gene frequency should beanalyzed for a number of generations over polluted and non-contaminated soils by a greenhouse or pilot field experiment(Pilon-Smits and Pilon, 2002). To further minimize the risk ofoutcrossing to wild relatives, the use of transgenics should beundertaken in consonance with classical breeding approaches.Transgenic plant species should be chosen that have no compatiblewild relatives, male-sterile transgenics may be bred, and the plantsmay be harvested before flowering (Pilon-Smits and Pilon, 2002).Field trials with transgenic poplars were carried out in formercopper-mining regions of Russia (Middle Urals, Swerdlovsk oblast)and Germany (Saxonia Anhalt, district Mansfelder Land) to assessthe biosafety risk of transgenic poplars developed for the remedi-ation of contaminated soils by elucidating the stability of thetransgene under actual field conditions and the possibility ofhorizontal gene transfer to microorganisms present in the rhizo-sphere. Preliminary results showed that the transgenic poplarswere genetically stable with no indications of any impact on theenvironment (Peuke and Rennenberg, 2005).

10. Conclusion

Phytoremediation is an environment friendly green technologyinvolving living plants which offers a cost-effective means forcleaning metal-contaminated soils. In order to exploit its fullpotential, a comprehensive understanding is needed on as to howmetal uptake, transport, and trafficking across plant membranesand distribution, tolerance, sensitivity, etc., take place underdifferent environments (Aruna Kumara, 2011). The technology ismore applicable to soils or ores that cannot be economicallyenriched by traditional mining. Biotechnology would enable the


identification of new genes to tackle the issue of environmentalcleanup. Several crop species are being researched upon and awaitincorporation of such genes not only for cleaning up the heavymetals but also to extend the area under cultivation. The challengefacing phytoextraction should be faced jointly by scientists, envi-ronmental engineers and science administrators to prove thetechnology’s efficacy at pilot sites. It is incumbent upon researchersto conduct basic laboratory work as well as follow new approachesfor identifying and solving diverse scientific issues posed by metalphytoextraction.

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