CA2249353A1 - Method for hyperaccumulation of metals in plant shoots - Google Patents

Abstract

The present invention provides methods by which hyperaccumulation of metals in plant shoots is induced by exposure to inducing agents. In preferred embodiments, manipulations that increase availability of metals to the plant are employed prior to application of the inducing agent. Effective inducing agents include conditions of low pH, chelators, herbicides, and high levels of heavy metals. Other phytotoxic agents are also useful. Application of multiple inducing agents results in synergistic effects.

Description

CA 022493~3 1998-09-21 SrHOD FOR HYPERACCUhnULAllON OF ~nETALS nN PLANTSHOOTS
Background of the Invention Activities in the Industrial Age have resulted in the deposit of high levels of many metals in certain sites, to the point that human life is seriously threatened. Metal-production activities, such as mining or smelting, as well as the ubiquitous use of metals, have created many sites where toxic metals have become concentrated in soils.
Although the problem has been recognized for many years, and much effort has been expended on methods to remove the metals, existing techniques are cumbersome, expensive, and invasive.
In recent years, efforts have been made to utilize metal-accllm~ ting plants to remove cont~min~ting metals from sites (see, for example, Baker et al. New Scientist 1603 :44, 1989; Chaney et al. in Land Treatment of Hazardous Wastes ed. by Parr et al., Noyes Data Corp: Park Ridge, pp 50-76, 1983). ~here are many advantages to usingplants for remediation, including lower costs, generation of recyclable metal-rich plant residue, applicability to a range of toxic metals and radionuclides, minim~l environmental disturbance, elimin~tion of secondary air or water-borne wastes, and public acceptance.
Unfo~ aLely, many plants accumulate metals to a concentration below the concentration of the metal in the soil, so that accumulation into the plant actually increases, rather than decreases, the amount of metal-conL;~ t~d m~teri~l.
Furthermore, most of the known metal-accumulating plants are slow-growing, smalland/or weedy plants that produce low biomass (see, for example, Baker et al. supra), so that even if the plants concentrate metals effectively, they cannot remove large amounts of metal from the soil. Also, most plants that accumulate metals collect the metal in CA 022493~3 1998-09-21 WO 97~4714 PCTrUS97/0495 their roots rather than into their above-ground shoot portions. In fact, it is generally accepted that most plants do not accumulate significant levels of heavy metals into their shoots. Since metal accumulated into plant roots cannot be removed from the site until the plant roots themselves are harvested, standard phytoremediation protocols require that the roots be harvested, an expensive and complicated process.
There remains a profound need for improved methods of reme~ ting metal-cont~min~te-l sites.

Summaly of the Invention The present invention provides a method for in-luçing plants to hyperaccumulate metals into their shoots. The invention therefore provides a novel and highly advantageous method for phytoremediation of metal-cont~min~te(l sites, as plant shoots can readily be harvested and removed from the site. The present invention conccn~l~ales metals in a readily disposable biomass to levels higher than the concentration of metal in the soil and thereby greatly reduces the weight of co~ tec~ m~t~ri~l that must be disposed. An additional benefit of the present invention, as compared with otheravailable techniques, is that the soil is cleaned rather than removed, and therefore remains available for use by the owner.
In p,cre.led embodiments of the present invention, a cultivar is planted in a metal cont~min~ted environment, the environment is manipulated so that availability of the metal in the environment to the plant is increased, the plant is allowed to take metal up into its roots, and the plant is then exposed to an inducing agent under conditions and for a time sufficient for the plant to hyperaccumulate metal in its shoots. Preferred CA 022493~3 1998-09-21 plants for use in the present invention include members of the family Brassicaceae, and particularly those of the genus Brassica. Other p~er. Il~d plants include members of the cucurbitaceae family, particularly pumpkins. Preferred inducing agents include chelating agents, organic acids, soil acidifiers, herbicides, and high concentrations of heavy metals.
The present invention provides an improved method for removing metal from an environment by cultivating a plant therein, in which the improvement comprises exposing the plant to an indllcing agent under conditions and for a time sufficient for the plant to hyperaccumulate metal into its shoots to a levels higher than it would if it were not exposed to the in~ cing agent.
The invention also provides a method for identifying agents that act to induce hyperaccumulation of metal into plant shoots. According to the present invention, a plant is grown in a metal-col ~tA~ d environment, is exposed to a potential in~lucin~
agent, such as a chemical or physical stress, and is analyzed to det~rmine the level of metal it accumulated into its shoots. Desirable inducing agents according to the present invention are those that stim~ te a plant to ~ccumlll~t~ more metal after exposure to the agent than it does without such exposure. Preferably, the plant is induce to ~ccum~ t~
at least twice as much metal in its shoots after exposure to the agent than it does without such exposure.
Brief Description of the Drawings Figure l is a bar graph showing the effects of EDTA on lead accumulation in roots and shoots of a Brassica juncea cultivar.

CA 022493~3 1998-09-21 W O 97/34714 PCTrUS97/04956 Figure 2 is a bar graph showing the effects of acidification on lead accumulation in roots and shoots of a Brassica juncea cultivar after acidification to pH 3.5.Figure 3 is a bar graph showing the combined effects of EDTA and acidification on lead acc--m~ tion in roots and shoots of a Brassica juncea cultivar.
Figure 4 is a bar graph showing the combined effects of EDTA, acidification, and an herbicide on lead accumulation in roots and shoots of a Brassica juncea cultivar.
Figure 5 is a bar graph showing the combined effects of EDTA and an herbicide on metal accumulation in roots and shoots of a Brassica juncea cultivar; the data demonstrate hyper~cc~lm~ tion of cadmium, copper, nickel, lead, and zinc.
Figure 6 shows the induction of lead hyperaccumulation into shoots of a Brassica juncea cultivar after exposure to high levels of a heavy metal.
Figure 7 is a bar graph showing the effects of certain soil amendments on uranium solubility in con~ te~l soil.
Figure 8 is a bar graph showing the effects of certain soil ~men-lments on soil uranium desorption and uranium hyperaccumulation in Brassica juncea shoots.
Figure 9 is a bar graph showing the ability of citric acid to induce uranium hyperaccllm~ tion into shoots of various B. juncea cultivars.
Figure 10 shows the ability of citric acid to induce ù~ hyperaccumulation in a variety of different plant species.
Figure 1 1 shows the time-dependent kinetics of uranium accumulation by a B.
juncea after application of citric acid to UldlliUIIl-CO~ .; "~te~ soil.
Figure 12 shows the synergistic effect of acidification and herbicide application in inducing uranium hyperaccumulation into plant shoots.

CA 022493~3 1998-09-21 Detailed D~ lion of Preferred Eml)o~ r e--ts of the Invention Metal hyperaccumulation according to the present invention occurs when plants are in~lucecl by application of an "in~ cing agent" to slccl-m~ te high levels of metals in their shoots. As noted above, the prior art teaches that plants do not typically transport significant levels of metals into their shoots (see, for example, C-mningh~m et al.
Bioremediation of lnorganics, Battelle Press, Columbus-Richland, 1995, p. 33-54). The present invention provides novel methods for increasing metal transport into plant shoots.
The present invention identifies a variety of useful inlluçing agents that stimulate hyper~ccllmlll~tion of metals in plant shoots. Generally, the present invention teaches that phytotoxic subst~n~es are useful in~ .ing agents. Without wishing to be bound by any particular theory, we propose that phytotoxic sub~ ces induce metal hyper~ccllml-l~tion by disrupting the plant metabolism in a way that overrides natural safety mech~ni~m~ that would otherwise operate to block transport of metal into shoots.
We note, however, that our theory does not suggest that the induction of metal l.dns~Jol I
described herein is exclusive of continued uptake of metal into plant roots. That is, metal uptake into plant roots probably c~ntin~ and may even be enhanced, during the induction period. We focus on the ll~ls~ l aspect primarily because it is clear that induction of hypeMccllmul~tion according to the present invention results in accumulation of significantly higher levels of metal in plant shoots than would be observed in the absence of such induction. Thus, whatever effects the inducing stimulus may (or may not) have on metal uptake into plant roots, transport into shoots is clearly enh~nce~l CA 022493~3 1998-09-21 W O 97/34714 PCT~US97/04956 Consistent with our model, we note that healthy plants, not exposed to inducing agents, typically do not accumulate significant levels of metals into their shoots. Also, metal accl]m~ tion into plant shoots has dramatic negative effects on plant growth. In the present invention, the negative effect on plant growth can be largely or almost S totally avoided by delaying the application of the inducing agent until the plants have accumulated a desirable amount of biomass. Then, because once the stimulus is applied, transport of metal into shoots is quite rapid, the metal-cont~ining shoots can be harvested without delay.
Thus, according to the present invention, selected plants are cultivated in an environment, typically soil, that is cont~min~ted with metal. After a period of plant growth, plants are induced by exposure to one or more inducing agents to hyperaccumulate metals into their shoots. An "in~ucing agent", according to the present invention, is any tre~tm~nt that, when applied to a plant or the soil, intluce5 the plant to accumulate more metal in its shoots than it would accumulate in the absence of the tre~tmtont Preferably, the plant is induce~ to accumulate at least about twice as much metal in its shoots as it would in the absence of the treatment.
For the purposes of the present invention, a plant is considered to have "hyperaccumulated" a metal in its shoots when, in response to an in~ cing agent, it has i) achieved a metal concentr~tion in its shoots (~g metal/g dry weight shoot mass) that is higher than the concentration of metal in the soil (mg/kg soil or mglL solution); and/or ii) ~ccllm~ ted at least about l O00 ~lg of metal per gram dry weight of shoot mass.
Preferably, the plant has achieved a metal concentration that is at least about two-fold higher than the concentration in the soil, and/or has accumulated at least about 3000 llg CA 022493~3 1998-09-21 of metal per gram dry weight of shoot mass. It will be appreciated that the goal is to induce plants to take up sufficient metal to reduce the metal concentration in the soil.
The absolute amount of metal accllm~ te~l according to the present invention depends on the type of metal being accumulated. For ex~mpl~, lead has proven to be a particularly difficult metal for plants to transport into their shoots (see, for example, Clmningh~m et al. Bioremediation of Inorganics, Battelle Press, Columbus-Richland, 1995, p. 33-54). According to the present invention, lead is preferably accumulated to at least about 3000 ~lg/g d.w. shoot mass, more preferably to at least about 4000 llg/g d.w. shoot mass, and most preferably to at least about 6000 ~g/g d.w. shoot mass (see Examples).
Hyperaccumulation according to the present invention is enhanced by procedures that increase the availability of metals in the soil (e.g., by increasing metal solubility) to plants cultivated therein. Increases in metal availability result in increased levels of metal ~ccumnl~tion into plant roots, which in turn results in increased levels of metal transport into plant shoots. Hyperaccumulation according to the invention is also enhanced by procedures that reduce metal pl~ci~ilaLion at and/or within the plant roots, as such precipitation limits the supply of metals available for 1~ l into shoots.
Accordingly, p~er.,lled embo~im~nt~ of the present invention involve manipulations that increase metal availability in soil and/or that inhibit metal p.ecipit~lion. In fact, some of the inducing agents specifically discussed herein, notably acids and metal complexing ager~- ("chelators"), act both as inducing agents and as promoters of metal solubility in the soil and/or ~,vithin the plant.
The present invention also demonstrates that combinations of in-lucing agents, CA 022493~3 1998-09-21 W O 97134714 PCT~US97104956 applied simultaneously or with intervening time periods, often have synergistic effects on metal accumulation. In prcr~lled embotlime~t~ ofthe invention, plants are exposed to a first manipulation that increases metal availability (e.g., by employing a first inducing agent that itself increases metal availability and/or by taking additional steps to enh~nce availability, as is ~ c~ ed below), and then to a second manipulation comprising application of an inducing agent that stimulates metal transport to the shoots. For example, we have found the application of low pH and/or a chelating agent as a first in~ucing agent, followed by a delay period and application of herbicide as a second inducing agent, results in very high levels of métal hy~ ccllmulation. It is particularly plc:re~led that plants be cultivated to high biomass prior to exposure to the first or second manipulations, in order that a large volume of plant tissue is available for metal accllmul~tion. It may also be desirable, however, for accumulation to be induced prior to 1ermin~tion of plant growth.
In the following sections, we present more thorough discussions of particular aspects of, and considerations relevant to, the present invention.

Plants Plant members that can be used in accordance with the present invention include any plant that is capable of being in(luçed to hyperaccumulate heavy metals by the methods described herein. Specifically, any plant that can be inrluçe~l to hyperaccumulate into its shoots a metal to a concentration greater than the corresponding concentration of metal in the growth media (soil) to be treated is useful in the practice of the invention.

W O 97/34714 PCTAUS97tO4956 Of course, not all plants can be in(lured to accumulate high levels of heavy metals in their shoots according to the present invention. In fact, even within a given plant species, not all cultivars will show the desired hyperaccumulation activity.
However, one of ordinaly skill in the art will readily be able to identify inducible plants ~ 5 by following the procedures set forth herein, in combination with known screening strategies (see, for example, Kumar et al., Environmental Science and Technology Vol.
29, No. 5, 1995). Any plant that, when cultivated in a metal-cont~min~t~d soil and exposed to an inducing agent as described herein, hyperaccumulates metal in its shoots to a greater extent than it would in the absence of the inducing agent is desirable.
Preferably, the plant is capable of acc-lm~ ting metal in its shoots to a concentration above that of the metal in the soil in l~ollse to the inducing agent. Preferred plant members for use in the present invention, in addition to being capable of hyperaccllm~ ting metal in their shoots to a concentration higher than that in the soil, have one or more of the following charactçri~tics:
(a) An ability to produce several crops per year. Plants that can produce several crops per year can remove greater quantities of metal from a given cont~min~ted site because the volume of biomass produced over a growing season with such a plant is greater than that for a single crop.
Because the amount of metal removed depends on the m~thprn~tical product of two factors- (1) the unit uptake of metal per unit of shoot weight (i.e, the concentration), and (2) the amount of harvestable biomass with said metal concentration- plants that produce more harvestable biomass are more likely to remove larger arnounts of metal CA 022493~3 1998-09-21 from the site.
(b) An ability to adapt for growth in various climates and soil conditions.
Use of adaptable plants takes advantage of the total know-how obtained ~vith a given plant species insofar as its agricultural and metal S ~ccl-m~ tion response is concerned. Thus, a particularly useful species about which much is known becomes even more useful and valuable insofar as its effectiveness applies in varying climatic and soil con lition~;
(c) An ability to adapt to modified conventional agricultural practices.
Plants that respond to conventional agricultural practices are ~,er~llcd for the present invention in~mnr.h as they can be easily cultivated and stimulated to produce vigorous root and shoot growth under intensive agricultural practices (i.e., Illechallical tillage, irrigation, fertilization, high plant populations). Particularly ~ f~ d are plants that can be adapted for use on co~ ed soils that require extremely intensive agricultural practices to produce vigorous growth in the face of soil conditions, such as shallowness, high gravel content, poor drainage, high salinity, or severe compaction, that are norm~l~y adverse to good growth;
(d) An ~m~ hility to genetic manipulation by mutagenesis and/or gene transfer. Plants amenable to genetic manipulation may be used to provide m~t~ 1 for genetic transformations to incorporate into other plants one or more characteristics desired for the practice of the present invention. Alternatively, plants amenable to genetic manipulation may CA 022493~3 1998-09-21 act as receptors of genetic transformations to develop or improve desired characteristics, thereby becoming useful (or more useful) in the present invention.
(e) An ability to grow to high biomass. Other characteristics being equal, plants that produce large amounts of biomass remove more metal from the soil in a given crop. It will be immediately recognized by those skilled in the art that selection solely by the criterion of volume of biomass produced is inappropriate because the other factor affecting the amount of metal removed in a crop - namely, the metal concentration in harvested shoots - will, like biomass production, vary from plant to plant.
Further, we have found in some of our ~xpc.;~ lion that prolonging the time interval before application of a given stimulus to, for example, applying the stimulus after the plant begins to s~- esce, may indeed result in greater biomass generation, but at the expense of a decrease in the amount of metal which can be concentrated into plant shoots.
Among the plants that are preferred for use in accordance with the present invention are those ~lesign~tecl herein as "crop members". "Crop members" are those plants that are grown primarily as either vegetative sources (e.g. as vegetables, forage, fodder, and/or condiments), or oilseeds. Crop members are preferred in the practice of the present invention prim~rily because they tend to produce large amounts of biomass.
Also plerell~d are "crop-related" members, which herein are defined as those plants that have potential value as a crop and/or as donors of agronomically useful genes to crop members. Thus, crop-related members are able to exchange genetic material CA 022493~3 1998-09-21 W O 97~4714 PCTAJS97/04956 with crop members, thereby permitting breeders and biotechnologists to perform interspecific (i.e., from one species to another) and hl~ eneric (i.e., from one genus to another) gene transfer, according to known techniques (see, for example, Goodman et al. Science 236:48, 1987, incorporated herein by reference).
S Particularly ~ ed plants for use in the practice of the present invention are members of the Brassicaceae family, preferably crop and/or crop-related members.Preferred members of the Brassicaceae family include, but are not limited to plants of the genera Brassica, Sinapis, Thlaspi, Alyssum, and Eruca. Particularly preferred are Brassica species B. juneea, B. nigra, B. campestris, B. carinata, B. napus, B. oleracea, and cultivars thereof. An especially useful B. juncea cultivar is number 426308.pnmpkin~ are also plcfelled plants for use in the practice of the present invention.
It should be understood that desirable plants for use in the present invention include those that have been mutagenized and/or genetically engineered (e.g., interspecific and/or ~ lic hybrids). Methods for mutagenizing plants are well known in the art (see, for example, Konzak et al., Tntern~tional Atomic Energy Agency, Vienna, 1972, pg. 95, incorporated herein by l~felence). Plants for use in the present invention can be genetically manipulated using known transformation techniques or using sexual and/or asexual (i.e., somatic) hybridization techniques. Hybridization techniques are well-known in the art, and have been employed, for example, to transfer agronomically important traits from related species to crop Brassicas (see, for example, Salisbury et al. Genet. Life Sci Ad~. 22 8:65, 1989, incorporated herein by reference).

CA 022493~3 1998-09-21 W O 97~4714 PCTAJS97/04956 Metals The present invention provides methods that are useful for the remediation of a wide variety of cont~min~ting materials. Accordingly, the term "metal" as used herein refers to metals (both stable and radioactive, both ionic and non-ionic forms), mixtures - 5 of metals, and combinations of metals with organic pollutants.
Metals that can be accumulated according to the present invention include antimony, arsenic, barium, beryllium, cadmium, cerium, cesium, chromium, cobalt,copper, gold, indium, lead, m~ne~nese, mercury, molybdenum, nickel, palladium, plutonium, rubidium, ruthenium, selenium, silver, ~L~ , tech~ nl~ thallium, thorium, tin, vanadium, uranium, yttrium, zinc, and combinations thereof.
Common organic pollutants relevant to the present invention include benzene or other aromatics, alkyl benzyl sulfonates (detergents), polycyclic hydrocarbons, polychlorinated biphenyls (PCB's) and/or halogenated hydroc~bo,ls (e.g.
trichloroethylene).
lS One advantage of the present invention is that the rapid induction of metal transport from roots to shoots allows plants to be utilized to acc--m~ te metals that have profound negative effects on plant viability. Of course, standard cultivation techniques teach the desirability of promoting plant viability. The only metals whose uptake is typically recommended are those that are e~eenti~l for plant growth (molybdenum,copper, zinc, m~ng~neee, iron; see Taiz et al., Plant Physiology, Ben~lnin/Cnmming~
Publishing Company, Inc., Redwood City, CA, pp. l 07-l 09, l 99 l ), and those only to relatively low levels. Prior art references, and indeed common sense, teach that it is undesirable, if not impossible, to use plants to take up metals that are poisonous to the CA 022493~3 1998-09-21 W O 97/34714 PCT~US97/04956 plants. The present invention, however, provides induction stimuli that trigger rapid metal transport, so that detrim~nt~l effects of metal accumulation are minimi7~d prior to induction. Thus, the present invention provides a novel method for the ~cc~ tion by plants of metals that are not essçrlti~l to, and/or are clçtnment~l to, plant growth. The present invention is particularly useful, and fills a void in existing techniques, because soils to be remediated are typically cont~min~ted with phytotoxic metals. Examples of metal cont:~min~ntc that are the primary toxic components of cont~min~ted sites are:
lead, chromium, arsenic, zinc, copper, cadmium, and nickel. Radioactive metals (e.g., uranium, plutonium, etc.) are also common in certain sites.
According to the present invention, lead is preferably accllm~ tecl to at least about 3000 ~g/g d.w. shoot mass, more preferably to at least about 4000 ,ug/g d.w. shoot mass, and most preferably to at least about 6000 ',lg/g d.w. shoot mass; zinc ispreferably ~ccllm~ ted to at least about 1000 ,ug/g d.w. shoot mass, and more preferably to at least about 2000 llg/g d.w. shoot mass; copper is preferably ~cc.lm~ tecl to at least about 1000 llg/g d.w. shoot mass, and more preferably to at least about 2500 ~lg/g d.w. shoot mass; cadmium is preferably accumulated to at least about 500 llg/g d.w. shoot mass, and more preferably to at least about 1000 ~g/g d.w. shoot mass;
nickel is preferably accumulated to at least about 200 llg/g d.w. shoot mass, and more preferably to at least about 500 ~g/g d.w. shoot mass (see Example 7). Uranium is preferably accumlll~ted to at least about 10 llg/g d.w. shoot mass, more preferably to at least l ~out 1000 ~lg/g d.w. shoot mass, and most preferably to at least about 4000-6000 g/g d.w. shoot mass.

CA 022493~3 1998-09-21 W O 97/34714 PCT~US97/04956 Metal-Co~ in~ Environment The metal-colltSI~ g environment in which plants are indllced to hyperacc-lmul~tç is not int~n-led to limit the scope of the present invention. That is, as long as the environment can sustain growth of the selected plants, it is suitab}e for the purposes ofthe present invention. Metal-c~ i,\g environments can range from purely aquatic environments with varying degrees of water saturation, organic matter content, mineral content, etc. to well-drained soils. Thus, the term "soil", as used herein, includes a wide variety of physical types and chemical compositions.

Plant Cultivation Various techniques for plant cultivation are well-known in the art (see, for example Canola Growers Manual, Canola Council of Canada, 1984, incorporated herein by ler~ ce). Plants can be grown in soil, or alt~ tively can be grown hydroponically (see, for e~mple, U.S. Patent No. 5,364,451; U.S. Patent No. 5,393,426; U.S.S.N.08/252,234; U.S.S.N. 08/359,811, U.S.S.N. 08/423,827; and U.S.S.N. 08/443,154, each of which is incol~,o~ d herein by reference).
Whereas the goal of cultivation in an ordinary crop plant for typical agricultural use is to m~imi7e the crop yield, the goal when practicing this invention is to increase in an lm~ Li~ted fashion the amount of above-ground biomass prior to induction.
That is, the biomass of impoll~lce to the effectiveness of the invention is the undi~l~llliated amount of biomass produced, in contrast to, for example, in corn, the more typical desire to achieve the maximum yield of edible m~teri~l. It also should be recognized, as elaborated above, that maximum crop yield per se should not be the sole CA 022493~3 1998-09-21 W O 97~4714 PCT~US97/04956 selection criteria, because it must be balanced with the concentration of metal in shoots upon accurnulation.
The optimal amount of time that a plant should be cultivated before application of the inducing stimulus according to the present invention will vary depending on the type of plant, the metal being accumulated, and the character of the environment in which the plant is being grown. For example, where Brassica juncea is being utilized to accumulate lead, it is generally desirable to cultivate the plants for at least three weeks, and preferably four to six weeks, after emergence of the plants before applying the induction stimulus (see, for example, Examples 2 and 5) Soil Manipulation As mentioned above, it is often desirable in the practice of the present invention to take steps to increase the availability of metal in the soil to the plant, and/or to reduce the likelihood of metal precipitation at or in the plant roots. The overall goal, of course, is to have the largest possible amount of metal taken up into the plant roots and available for transport into the shoots.
The term "increase the availability of metal", as used herein, refers to rendering metals in an environment more amenable to plant root uptake, and/or to subsequent shoot transport, than they would be in the absence of the manipulation. Manipulations that can increase the availability of metal to plants include, for example, (i) addition of chelators to the soil; (ii) tilling of soil to bring metals cont~ining soil into contact with the plant root zone; (iii) decreasing pH of the metal-cont~ining environm~nt, for example by adding an effective amount of an organic or inorganic acid (such as, for CA 022493~3 l998-09-2l WO 97/34714 PCT~US97/04956 example, nitric acid, acetic acid, and citric acid), or by adding to the environment a compound, such as ammonium sulfate, that will be metabolized by the plant roots (and/or by associated bacteria or other component(s) of the rhizosphere) in a manner that produces protons and thereby reduces the soil pH (see, for example, U.S.S.N.
08/252,234, incorporated herein by reference; see also Example 10).
As noted above, certain of these manipulations that increase soil availability (e.g., addition of chelators and acidification of soil) also can serve as inducing agents that stimulate metal transport to the shoots. The effects of these agents on metal transport are distinct from their effects on metal availability (see below).
Given that it is often useful to increase the availability of metals to plants, it is also typically desirable to avoid taking measures that would reduce such availability.
For example, when delivering phosphate fertilizers to plants, it is typically desirable to employ techniques, such as spot or foliar fertilization, that will minimi7P formation of insoluble metal phosrh~tec.
Induction Stimulus Any of a variety of agents applied to the soil and/or to plant foliage can be used to induce metal hyperaccumulation in plant shoots, in accordance with the present invention. Desirable inducing agents, used either alone or in combination, include metal chelators, organic and inorganic acids, herbicides, plant growth regulators, and other phytotoxic compounds.

CA 022493~3 l998-09-2l W O 97/34714 PCT~US97/04956 Chelators We have observed that exposure to a chelating agent can effectively induce metal hyperaccumulation in plant shoots. In particular, we have found that exposure to ethylenetli~minetetraacetic acid (EDTA) and other çhel~tinE agents well known to those skilled in the art induces hyperaccumulation of lead into shoots of B. juncea cultivars (see Examples 1 and 2).
As discussed above, chelators such as EDTA improve metal solubility in the soil, and thereby increase availability of the soil metals to the plant. This increase in metal solubility pre~ ably increases the amount of metal accumulated into the plant.
However, the evidence presented in Examples 1 and 2 shows clearly that EDTA has an effect on metal accumulation into shoots that is beyond any effect it has on metal availability because the observed hyperaccumulation of lead into plant shoots does not increase linearly with EDTA concentration, as would be expected for a solubility effect.
Rather, lead uptake increases dramatically above a threshold level (greater than about 0.3 mmol/kg at pH 5.1 and greater than about 1.0 mmol/kg at pH 7.5 in Example 2).
Thus, we have demonstrated that EDTA induces hyperaccumulation of lead into plant shoots by stim~ ting l,a,ls~,olL of root-acc-lm~ ted material.
Various chelating agents other than EDTA are known in the art and have been used in plant cultivation as a source of micronutrients or to enhance solubility of essential metals. The present invention teaches that, in addition to these known uses of chelators in plant cultivation, chelators are also useful to induce metal hyperaccumulation into plant shoots if applied in the manner described. One of ordinary skill in the art will appreciate that other metal chelating or complexing agents CA 022493~3 1998-09-21 W O 97/34714 PCT~US97/04956 such as, for example, ethylene glycol-bis(~-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTPA), trans-1,2-tli~min-~cyclohexane-N,N,N',N;-tetraacetic acid (CDTA), N-hydroxyethylethylene-~ rninetriacetic acid (HEDTA), nitrilotriacetic acid (NTA), citric acid, salicylic acid, and malic acid, can desirably be used in accordance with the present invention, and can follow the teachings of the present specification to screen and identify particular chelators and conditions that may be plc;rel~d for specific applications.
With this in mind, we note that an extensive literature exists on the selection and specificity of synthetic and natural chelate binding affinities for specific cations in soil and water systems (see Lindsay, Chemical Equilibria in Soils, Wiley Interscience, New York, 1979; Norvell, Soil Sci. Soc. Am. J. 48:1285, 1984; Norvell, Micronutrien~s in Agriculture, Second Edition, Soil Science Society of America Book Series No. 4, Soil Science Society of America, Madison, WI, 1991; Sommers et al., Soil Sci. Soc. Am. J:
43:39, 1979). In addition, several computer software packages have been developed to aid the evaluation of solid and solution phase equilibria in the soil system in the presence of added chelates (Loeppert et al. Chemical Equilibrium and Reaction Models, Soil Science Society of America Special Publication Number 42, Soil Science Society of America and Arnerican Society of Agronomy, Madison, WI, 1995).

Acidification Example 3 reports our finding that exposure of B. juncea plants to pH 3.5 in solution culture induces hyperaccumulation of lead into plant shoots. We also present data in Example 4 demonstrating that the sequential ~mini~tration of an acid and CA 022493F,3 1998-09-21 EDTA induces higher levels of lead accumulation into B. j~ncea shoots than are inrl~lce~l by a(lmini~tration of either the acid or EDTA alone. Furtherrnore, Exarnple 5 demonstrates that a combination of acid and EDTA induces metal transport into shoots effectively in a field enviromnent. This finding is particularly significant because large-scale acidification of soil to pH 3.5 may well be impractical in soil sites. The data presented in Example 5 demonstrate that such large-scale acidification is not required.
Some level of acidification (we note that the quantities of acid used in Example 5 only slightly reduce the soil pH) is still valuable due to its synergistic effects when combined with another in-lucing agent such as a chelator.
Fx~mple 11 plcst~ our fin-linp~ that reduction of soil pH dramatically enhances accumulation of uranium into shoots of a wide variety of di~le.ll plants. As shown in Example 11, we found citric acid to be a particularly effective inducing agent, probably because of its dual abilities to i) acidify the soil; and ii) chelate the Ula~ n.
We note that standard techniques of plant cultivation in soils ~col...,-~n~l that pH
be m:~int~ine~l at about pH 5.5-7.0 for optimum growth of most crops. In fact, a large litel~ has developed that describes how best to treat different types of soil to ensure that a desirable pH is m~qint;qined (see, for example, Cornmercial Vegetable Production Recommendations, Reiners and Garrison, eds., Rutgers, State University of New Jersey, 1994, pp. 1~-27; "Agronomy of Canola in the United States", pp. 25-35 in Canola and Rapeseed, Production, Chemistry, Nutrition, and Processing Technology, ed. F. Shahidi, Van Nostrand Reinhold, New York, 1990, each of which is incorporated herein by reference).
Thus, according to the present invention, a soil pH greater than about 5.5 is CA 022493~3 1998-09-21 desirable in the initial cultivation stage during which most of the biomass is accllm~ t~l This initial cultivation stage is followed by a reduction in pH to induce metal accumulation. As described in the Examples, soil pH is preferably reduced to about pH 3.5, though less dramatic pH reductions are also desirable, especially when an additional inducing agent is employed. In fact, any acidification (either localized or general) ofthe soil-root system is expected to be beneficial to the induction mech~ni~m when used in combination with other inducing agents, regardless of its ability to stimulate induction in the absence of other inducing agents.
The principles exemplified by the data in F.Y~mples 3, 5 and 12 are, Gf course, not limited to the exemplified plants, nor to the precise cultivation and/or induction conditions described. For example, dirr~.en- pH ranges may be optimal for induction in different plants. One of oldin~y skill in the art can readily follow the teaching~ of the present specification to screen dirr~ l plants and conditions and identify those combinations that result in induction of h~ c~ tion into plant shoots.
Also, in Examples 3 and 5, solution pH was reduced by application of 1.0 N
HNO3. Alternate acidifying agents (such as, for example, acetic acid, ammonium acetate, ammonium sulfate, ferrous sulfate, ferrous sulfide, elemental sulfur, sulfuric acid, citric acid, ascorbic acid) can be used to reduce the soil pH (see, for example, Example 12). Also, soil pH can be reduced by addition of a metabolite that is processed by the roots or other element of the rhizosphere in a marmer that produces protons (see above). Plefe..~d acidifying agents are those that chemically or biologically degrade within days or weeks without leaving residual salts that may either result in an undesirable buildup of salinity (i.e., ammonium, chloride or sodium) or create a CA 022493~3 1998-09-21 potential environment~l hazard from le;~ching of the associated anions (i.e., nitrate from nitric acid). Particularly plefell~d acidifying agents include, but are not limited to, acetic acid, citric acid, or ascorbic acid.

Herbicides The application of selected herbicides to B. juncea plants grown in heavy metal cont~min~ted soil ~men-l~d with chelators in~ ced hyperaccumulation of metals into plant shoots (see Examples 5-7 and 12). Examples 5-7 demonstrate that several different commercially available herbicides can be used in accordance with the present invention to induce hyperaccumulation of metals into plant shoots; Fx~mple 12 shows that herbicides can act synergistically with acidification to induce metal hyperaccumulation. It is worth noting that, under the conditions of Examples 5-7, herbicides did not effectively induce hy~el~c~ tion in soil environments in the absence of an agent (e.g., acid or chelator) that increased metal availability to the plants (see Figure 4). Without wishing to be bound by any particular theory, we propose that metals first accumulate in the plant roots, and that the induction stimulus inrlllces transport to the plant shoots. Of course, one of ordinary skill in the art will recognize that, in a system where metal availability is not a problem (for example, in a hydl~pollic system), herbicides, and other in-lucing agents that do not also increase metal availability, may still effectively induce metal hyperaccumulation into plant shoots.
In ~lcr~ d embodiments of the present invention, reflected in the Examples, herbicides are applied as inducing agents only after the plants have first been exposed to an agent that increases metals availability (e.g., acid and/or chelator). Furthermore, in CA 022493~3 1998-09-21 many cases, a delay (e.g., 24 hours) is desirably imposed between the application of the treatment that increases metal availability and the application of the herbicide. The idea is to allow metals to accumulate in the roots during application of the treatment that increases metal availability, and then to induce transport of root-accumulated metal into the shoots by application of the herbicide. One particularly ple~ d embodiment of the present invention involves sequential application of EDTA and herbicide (e.g., Roundup~), with a delay in between. However, alternative embotliment~, in which herbicide is applied simultaneous with or even prior to application of other ~men~1ments, are also encompassed within the scope of the present invention. Particularly where the metal being ~rcl-m~ te~l is uranium, application of herbicide prior to (e.g., approximately 6 hours before) application of other ~m~n~lmenl~ is desirable.
Those of ordinary skill in the art will recognize that any of a variety of herbicides other than those specifically plesented in the Examples are useful inducing agents in accordance with the present invention. Preferred herbicide compounds have little or no residual soil activity and decompose quickly in the environment. Such er~ d compounds include commercially available formulations co.~ g, for example, glyphosate, 2,4-dichlorophenoxyacetic acid (2,4-D), 2-methyl4-chlorophenoxyacetic acid (MCPA), or maleic hydrazide.

Other agents Those of ordinary skill in the art will readily recognize that any of a variety of other agents can be used as inducing agents in the practice of the present invention.
According to our theory, any agent that disrupts plant metabolism in a way that CA 022493~3 l998-09-2l W O 97/34714 PCT~US97/04956 overrides the natural protective mech~ni~m~ that block root-to-shoot transport of phytotoxic materials will be effective in inducing metal hyperaccumnl~tion in plant shoots. Consistent with this hypothesis, we have found that high levels of heavy metals can also function as inducing agents according to the present invention (see Examples 8-9). Significantly, as can be seen with reference to Examples 8 and 9, hyperaccumulation is only in~ ced above a threshold level of metal.
The present invention therefore teaches that exposing plants to a physiological stress or phytotoxic substance (e.g. phytotoxic levels of metals or nutrients, low pH, osmotic stress, herbicide, etc.) or combination of such subst~ncçs, disrupts the plant's natural safety meçh~ni~m~ norm~lly involved in preventing uptake and/or transport of toxic substances into plant shoots and stimul~tes metal translocation from the roots to the shoots. Thus, according to the present invention, any agent with phytotoxic activity can be screened to test its ability to induce metal hyperaccllm~ tiQn in plant shoots according to the procedures described herein.
For example, any or all ch~l~ting agents (e.g., EDTA, EGTA, DTPA, CDTA, citric acid, salicylic acid, malic acid), soil acidifiers (e.g. acetic acid, ammoniurn acetate, ammonium s--lph~te, ferrous sulfate, ferrous sulfide, el~ment~l a sulfur, sulfuric acid, citric acid, ascorbic acid), phytotoxic levels of plant nutrients and trace elements (Fe, Mn, Na, Al, etc.) and cornmercially available herbicides (co~ g e.g., glyphosate, MCPA, maleic hydrazide) alone or in combination with one another, can be tested for their inducing capabilities, as can other chemical agents such as other toxins, detergents, enzyrnes, and plant hormones, or physical factors such as drought, extreme heat,ultraviolet radiation, and x-radiation. Also, any of these agents can be tested under CA 022493~3 1998-09-21 conditions of nutritional starvation, but starvation alone is not sufficient to induce metal hyperaccumulation into plant shoots.
Those of ordinary skill in the art can readily screen any or all of these materials for inducing capability according to the procedures described herein. Desirable agents are those that stimulate a plant to accumulate metal in its shoots to a level higher than the plant would accumulate in the absence of the agent. Preferably, the agent stimulates the plant to ~cl-m~ te at least about two-fold more metal in its shoots than the plant would do if not exposed to the agent, more preferably, the agent induces the plant to accumu}ate at least about 1 0-fold more metal in its shoots, still more preferably at least about 1 00-fold more, and most preferably at least about 1 000-fold more.

Harvesting Plant shoots into which metals have been hyperaccumulated in accordance with the present invention are harvested by any of a variety of standard techniques, such as swathing, chopping, or baling. Shoot harvesting from certain Brassica species, such as B. campestris, B. juncea, and B. nabus, in particular is routine (see, for example, Canola Growers Manual; Canola Council of C~n~ , 1984, incorporated herein by reference).
Often, in the practice of the present invention, it is desirable to produce multiple s-lccessive crops in a single growing season, in order to effect the greatest amount of metal removal on a given site. Thus, it is typically desirable to harvest shoots pn~nl~lly after the completion of the induction process, in order to waste no time in a limited growing season. The induction process is complete when leaves of the plant become desiccated and begin to drop. When it is desirable to prevent undue loss of metal-rich CA 022493~3 1998-09-21 W O 97t34714 PCTAJS97/04956 plant material, harvest should begin at the first sign of leaf desiccation and/or leaf drop, and should be completed without delay thereafter.

Examples EXAMPLE l: Inducing Hyperaccumulation of Lead by Addition of EDTA.
Materials and Methods:
Seeds of B. juncea cultivar 426308, obtained from the USDA/ARS Plant Introduction Station of Iowa State University, were cultivated hydroponically in open-ended 1.7 mL microcentrifuge tubes packed with 1 cm3 of vermiculite, so that the roots of cultivated plants extended into an aerated nlltrient solution of 1 g/L HydrosolTM
supplemented with Ca(NO3)2. During cultivation ofthe see-llin~s, six tubes supported by a floating styrofoam platform were placed in an 1 8L tray cont~ining 1 SL of solution.
Experiment~ were done in an enviro~ ent~lly controlled growth chamber at 25~C, 75%
relative hu~nidity, and a 16 hour photoperiod was provided by a combination of ine~n-lescçnt and cool-white fluorescent lights.
After three weeks, plants were rinsed in deionized water for 20 minutes and thenwere transferred to a container with 750 mL of metal-co~ te~l solution. Lead nitrate was used to obtain a 50 mg Pb/L solution. The solution concentration remained constant for the duration ofthe ~xpe. ;..,~nt. EDTA was added to ~ JL. ;~ent~l chambers to a concentration of either 0.2 mM or 1.0 mM, by addition of 0.5 M EDTA
stock -~lution. Plants were exposed to the lead-cont~min~ted solution, in the presence of EDTA, for 7 days, and then were harvested.
Plant roots and shoots were harvested separately, dried for 48 hours at 70~C in a - CA 022493~3 1998-09-21 W O 97/34714 PCTrUS97J04956 forced air oven, weighed, ground, and wet digested with nitric and perchloric acids. At least 4 rep}icates were used for each treatment.
The metal content of the extracted acid was deterrnined with a Fisons Direct Current Plasma Spectrometer, model SS-7.

Results and Discussion Results are presented in Figure 1. As can be seen, in the absence of EDTA, soluble lead in the solution is accumulated into plant roots, but is not transported to the shoots in appreciable amounts. By contrast, addition of EDTA results in high levels of accumulation in plant shoots, and a reduced amount of lead remains in the plant roots.
This phenomenon is not explained by EDTA's known ç~p~bility to solubilize metals in the soil solution, since the metal was already dissolved in the test solutions. The hyper~ccum~ tion of metal and transport to shoots is app~l~."ly related to the stress on the plant caused by EDTA, which has phytotoxic effects at high concentrations.
We note that the results presented in Figure 1 also show that lead accumulation into roots is increased in the presence of EDTA, evidencing an ability of EDTA to increase metal availability even in this hydroponic system. This finding s~ggestc that EDTA has effects not only on metal solubility in soil, but also on metal solubility on and/or inside plant roots, so that EDTA helps m~int~in the metal in a form (perhaps an EDTA/metal complex), that is suitable for shoot transport. The chelator may also act to bind calcium at the root surface, thereby reducing metal precipitation, and/or to increase membrane permeability, thereby allowing less restricted movement of metal into the root.

CA 022493~3 1998-09-21 W O 97/34714 PCTrUS97/04956 EXAMPLE 2: Addition of EDTA to Soil Materials and Methods:
A Sassafras Ap silt loarn soil was collected from the Rutgers University Horticultural Farm and amended with lead carbonate. The soil was limed to pH 5.1 or 7.5, and was fertilized with urea (150 mg N/kg), potassium chloride (100 mg K2O/kg), and gypsum (70 mg CaSOJkg). The soil was allowed to e4uilibrate for two weeks inthe greenhouse at saturation, air dried, and remixed before planting. The soil was placed in 8.75 cm diameter pots (350 g soil/pot) and planted with Brassica juncea (426308) seeds. Phosphate fertilizer was added as a spot placement 1 cm below the seeds at planting at the rate of 100 mg P205/kg. After see~lling emergence, the pots were thinned to two plants per pot.
Plants were grown for three weeks in a growth chamber with a 16 hour photoperiod and were given weekly fertilization treatm~nts of 16 and 7 mg/kg N and K, respectively. Three weeks after see~lling emergence, chelate (EDTA as a K salt) solutions were applied to the soil surface. The pots were placed in individual trays to prevent loss of amendments from leaçlling Following the chelate applications, the soil was irrigated to field capacity on a daily basis. The plants were harvested one week after the chelate treatment by cutting the stem 1 cm above the soil surface. The plant tissue was dried and analyzed for metal content by ICP as described previously in Example 1.

Results and Discussion:
Results are ~l~st;lll~d in Table 1:

W O 97/34714 PCT~US97/04956 Pb accumulation in B. juncea shoots after amendment of soil with EDTA.
Sassafras Ap soil (pH 5.1) EDTA PbConcentration mmol/kg in Shoots ~lg/g 0.3 917+221 0.5 3066+ 1362 1.0 6748 + 1842 2.5 8162 + 2501 5.0 11740 + 3802 7.5 15321 + 1491 Sassafras Ap soil (pH 7.5) 0.0 15 ~ 1 151.0 243~t35 2.5 1398 + 560 5 o 5590 ~ 1916 As can be seen, accumulation of lead into the shoots was not a linear response to 20the amount of EDTA added to the soil. This finding indicates that the effect of EDTA
was not due solely to the chelator's ability to increase metal solubility in the soil.

EXAMPLE 3: Tn-lucing Hyperaccumulation of Lead by Altering pH.
Materials and Methods:
25Seeds of Brassica juncea cultivar 426308 were obtained from the USDA/ARS
Plant Introduction Station of Iowa State University.
See-llin~ were cultivated hydroponically in open-ended 1.7 mL microcentrifuge CA 022493~3 1998-09-21 W O 97/34714 PCT~US97/04956 tubes packed with 1 cm3 of vermiculite, with roots exten~ing into an aerated nutrient solution [1 g/L HydrosolTM supplemented with 0.6 g/L Ca(NO3)2]. During cultivation of the see~llingsl six tubes supported by a floating styrofoam platforrn were placed in an 18 L tray cont~ining 15 L of solution. Experiments were done in an environmentally controlled growth chamber at 25 ~C, 75% relative humidity, and a 16 hour photoperiod provided by a combination of inc~nllesc~nt and cool-white fluorescent lights.
After three weeks, plants were rinsed in deionized water for 20 rninlltes and then transferred to a container with 750 mL of cont~min~te~l solution. Lead nitrate was used to obtain 50 mg Pb/L solution. Solution concentration r~m~in~l constant for the duration of experiment. Solution pH was adjusted to either pH 5.5 or pH 3.5 by addition of 1.0 N HNO3. Plants were exposed to the lead cont~rnin~ted solution, under the low-pH conditions, for 7 days, and then were harvested.
Roots and shoots were harvested separately, dried for 48 hours at 70~C in a forced air oven, weighed, ground, and wet digested with nitric and perchloric acids. At least 4 replicates were used for each treatment The metal content of the acid extract was ~letermined with a Fisons Direct Current Plasma Spectrometer, model SS-7.

Results and Discussion:
Results are presented in Figure 2. As can be seen, reducing the pH of the cont~min~te~l solution from 5.5 to 3.5 drarnatically changed the arnount of lead taken up by B. juncea shoots. Plants exposed to 50 mg/L lead solution at a pH of 3.5 accumulated 6 mglg lead, some 100 times the arnount taken up at a pH of 5.5. This CA 022493~3 l998-09-2l phenomenon cannot be explained by increased lead solubility, since the soluble lead rem~in~ at 50 mg/L during the entire experimental period at either pH level.

EXAMPLE 4: Synergistic Induction of Lead Hyperaccumulation by Exposure to a Sequence of Altered pH and EDTA
Materials and Methods:
Experiments were pelrolllled as described above in Example 3 except that, after the plants were exposed to the lead-co~ solution at the adjusted pH, EDTA was added. Four different reaction conditions were ntili7e(l a. Control: pH = 5.5, no EDTA addition b. pH = 5.5, EDTA added at 0.2 mM
c. pH = 3.5, no EDTA addition d. pH = 3.5, EDTA added at 0.2 mM
pH of the solutions was adjusted using a 1.0 N HNO3 solution. EDTA was added after pH adj.~tment using 0.5 molar stock solution. At least 4 replicates were used for each treatment.

Results and Discussion:
Results are presented in Figure 3. As can be seen, the combination of low pH ( 3.5) and EDTA application has a synergistic effect. The sequence of pH adjustment to 3.5 followed by a dosage of EDTA results in hyperaccumulation levels much higherthan the use of a single addition of EDTA or of acid . The lead concentration in dried CA 022493~3 1998-09-21 shoots of 1.7% and the corresponding bioaccumulation coefficientl of 340 achieved ~,vith the combination of pH 3 .5 and addition of EDTA are higher than any values reported in Examples 1-3.

EXAMPLE 5: Effect of AcidlChelator/Herbicide Sequences in Inducing Lead Hyperaccumulation in Field Trial Site Materials and Methods:
A field study was conducted at a site in Bayonne, NJ with Pb cont~min~te~l soil (1200 mg Pb/kg). Soil was fertilized with 150, 100, and 70 mg/kg of N, K20, and CaSO4, respectively. The surface soil (0-15 cm) was excavated and placed in lysimeters (48 qt ice chests). 65 kg of soil was placed in each lysimeter and the lysimeters were placed on the surface of the soil in the field. B. juncea seeds were planted and grown for 3 weeks before treatment application, EDTA and acetic acid were applied as 1 L
solutions to equal 5.0 rnmol/kg of EDTA and acetic acid. Herbicide treatments were applied 24 hours after the EDTA and acetic acid tre~tmlont~ using a 12.5% RocklandTM
(a mixture of Prometon and 2,4-D) solution to wet the foliage. Plants were harvested one week after treatment application. Root and shoot tissue was collected and washed to remove soil deposition before analysis.

Bio~rcl-m~ ti~n coefflri~nt = (~g metal uptake/g of dry shoot mass) divided by either (/lg metal in substrate/g dry weight of soil) or (llg metal in substrate/mL of solution), depending on whether the system is soil-based or hydroponic.

CA 022493~3 1998-09-21 W O 97/34714 PCT~US97/04956 Results and Discussion:
Results are presented in Figure 4. As can be seen, addition of acid in conjunction with EDTA results in synergistic induction of lead hyperaccurnulation in shoots. These findings are particularly significant because they represent an effect that S occurs in the field, at a cont~min~te~l site. These fin~ling~ therefore show that the present invention is practical for phytoremediation of cont~min~tecl sites.

EXAMPLE 6: Addition of Sequence of Chelator and Herbicide to Soil Materials and Methods:
Soil was prepared and plants were grown as described in Exarnple 2. After three weeks of growth, EDTA was applied to the soil solution at the rate of 2.5 mmol/kg.
Twenty-four hours after the chelate solutions were applied, herbicide solutions of Paraquat, Roundup~ (glyphosate), or Rockland~ were applied in various concentrations to wet the foliage. Plants were m~int~ined as described in Exarnple 2, and were harvested 7 days after the chelate application.
Results and Discussion:

Results are pl~s~ d in Table 2:

Pb accumulation in B. juncea shoots after arnenc~mPnt of soil with EDTA and herbicide HerbicideConcentration Pb Uptake %(v/v) llg/g Control (EDTA 0.0 1 178 ~ 579 alone) W O 97/34714 PCT~US97/04956 Paraquat 0.5 6216 + 1027 2.0 3099 + 344 5.0 3606 i 48 Rockland~ 12.5 4710 ~ 484 18.3 3955 + 457 3479 + 246 Roundup~ 1.5 6682 + 1461 5.0 4939 + 1004 4390~ 1216 As can be seen, sequential addition of EDTA alld an herbicide results in synergistic effects on induction of metal hyperaccumulation into plant shoots.

EXAMPLE 7: Effect of EDTA and Herbicide Applications on Induction of Hyperaccumulation of Various Metals from Conl;~ te~1 Soil.
0 Materials and Methods:
The Sassafras Ap soil was amended with oxide and carbonate forrns of Cd, Cu, Ni, Pb, and Zn and prepared as in Exarnple 2. Chelate solutions were applied with an herbicide application of 2,4-D as described in Example 5.

Results and Discussion:
Results are presented in Figure 5. As can be seen, the combination of EDTA
and an herbicide induced hyper~ccllmul~tion of a variety of dirr~lc~lL metals. The 2,4-D
herbicide inciuced hyperaccumulation of all metals.

CA 022493~3 l998-09-2l EXAMPLE 8: Inducing Hyperaccumulation by Exposure to High Concentrations of Metal Materials and Methods:
Twelve different species of plants as listed in Table 3 below were tested. SeedsS of Brassica species including B. nigra, B. olerace~, B. campestris, B. carinata, B.
juncea and B. napus were obtained from the Crucifer Genetics Cooperative, Madison, Wisconsin. Seeds of other plants were purchased from local seed markets.
See~llings were grown in a greenhouse equipped with supplementary lighting (16 h photoperiod; 24-28~C; see Kumar et al. Environ. Sci. Technol. 29:1232-1238, 1995, incorporated herein by reference). See~lling~ were grown for 10 days in acid-washed coarse sand and fertilized every two days either with full-strength Hoagland's solution or with 1 g/L HydrosolTM supplemented with 0.6 g/L Ca(NO3)2. Ten-day-old see-llin~
were transplanted (in sets of two) into 150 g dry weight (DW) of an acid-washed 1:1 (v/v) mixture of coarse sand and coarse Perlite placed in 3.5 inch round plastic pots.
The pots contained two different levels of lead- 62.5 mg/kg or 625 mg/lcg dry weight sand/Perlite. Each pot contained two see-lling~ At least four replicates for each metal concentration were used.
Every other day the plant leaves were fertilized with MiracleGroTM solution until most of the leaves were wet. Phosphates and sulfates were not used, to avoid precipitation of Pb and other heavy metals.
Plants were grown for 14-20 days. Shoots of metal-treated and control plants were harvested and washed thoroughly with running tap water. Plant tissue was cut into small pieces with scissors, dried for 2 days at 80~C and ashed in a muffle furnace at , W O 97/34714 PCT~US97/04956 500~C for 6 h. The ash was dissolved in a mixture of 2M HCl and I M HN O3. The metal content of the acid extract was determined with a Fisons Direct Current Plasma Spectrometer, model SS-7.

Results and Discussion Table 3 below compared the accumulation of Pb in shoots of the 12 species tested at two different levels of Pb.

Metal P l~tir~n in ~ hoots and bi~ a~ rr;. ~
Lead level of substlate sand/Perlite mixture mg/kg d y weight 62.5 625 Biomass l~oeff~- ~ Biomass Co~PffiriPnt llglg llg/g Brassicafuncea (L.) Czern. 30 0.5 10,300 16.5 B. nigra (L.) Koch 30 0.59400 15.0 B. ~ , .~ L. 30 0.57200 11.5 B. carinataA. Br. 40 0.64600 7.4 B. napusL. 30 0.53400 5.4 B. oleraceaL. 50 0.8 600 1.0 Helianthus annuus L. 5600 9.0 Nicotiana tabacum L. 800 1.3 Sorg~lum bicolor L. 300 0.5 ,47, ,.. ~I.c.shybridusL. 300 0.5 A. paniculataL. 400 0.6 Zea mays L. 200 0.3 This experiment, and the ex~e.;l~lents reported in Example 9 below, demonstrates that heavy metal can be used as an agent to induce hyperaccumulation of metals into plant shoots. As can be seen, induction does not occur unless the CA 022493~3 1998-09-21 W O 97/34714 PCT~US97/04956 concentration of the metal in the environment is above a threshold level.
Specifically, the data p~se~t~d above in Table 2 show that low levels of available lead (e.g., less that 62.5 mg/Kg in this Example) in the growth medium do not induce metal hyperaccumulation in shoots for any species tested; witness that concentrations of lead in shoots do not exceed 50 llg/g DW in plants exposed to 62.5 mg Pb/Kg, and the bioaccumulation coefficient for these plants remains below 1Ø By contrast, plants exposed to a ten-fold higher concentration of lead in the environment (here 625 mglKg) show completely unexpected, and di~,opollionately high levels of lead accumulation in plant shoots. The level of lead uptake observed- IO as much as 1%
of shoot dry weight- is unprecedent~d We note that most of the Brassica species we tested are inrluced to hyperaccum~ te lead into their shoots after exposure to high lead levels. Among the other species tested, only sunflower (Helianthus annuus) and tobacco (Nicotiana tabacum L.) had bioaccumulation coefficients above 1Ø Sunflower, like many of the 1~ Brassicas, is an excellent plant for hyperaccumulation and thus phytoextraction.
The finding that high concen~ ions of heavy metals can serve as inducing agents to stiml-l~te metal hyperaçcumul~tion into plant shoots, when taken in light of the other fin-lings reported in the present application, may influence decisions regarding strategies for phytoremedi~tion of cont~min~ted sites. For example, as discussed above, we have found that multiple inducing agents can act synergistically to stimulate metal hype ccumulation into plant shoots. Thus, one of ordinary skill in the art will recognize that the levels of, for example, acid or chelating agent that can desirably be employed to induce metal hyperaccumulation at a site may well vary depending on the CA 022493~3 1998-09-21 concentration of metal already present in the site, as the metal itself may contribute to the induction effect. Furthermore, it may well be the case that high levels of one type of metal can induce plants to hyperaccumulate other types of metal that are not present in such high concentrations in the environment. Regardless, the present invention teaches S that high levels of heavy metals can act as an inducing agent to stimulate metal hyperaccumulation into plant shoots.

EXAMPLE 9: Induction of Hyperaccumulation by Varying Lead Levels Materia~s and Methods:
0 B. juncea cultivar 182921 was employed in experiments in which plants were grown hydroponically in a manner similar to that described above in Example 1. Roots of 17-day-old see~llings were exposed to 400 mL of aqueous solution Co~ 'i"g varying amounts of lead (0, 6, 22, 47, 94 or 188 mg Pb/L). After an additional 14 days, plants were harvested. Metal content of plant parts was analyzed using the procedures detailed in Example 8.

Results and Discussion:
Results are presented in Figure 6. As can be seen, the concentr~tion of lead accumulated in B. juncea roots increased with increasing solution concentration, though some decline in rate was observed when lead was present in the solution at concentrations above about S0 mg/L. By contrast, the concentration of lead accumulated in B. juncea shoots did not increase significantly until the concentration of lead in the solution approached 100 mg/L. At the highest concentration of lead tested CA 022493~3 1998-09-21 W O 97/34714 PCT~US97/04956 (188 mg/L), lead levels in shoots reached about 1.6%.
The results presented in Figure 6 confirm the fin~lin~ reported in Example 8, that lead hyperaccumulation into plant shoots is only induced by exposure to heavy metal when the metal is present in a concentration above some threshold value.
s EXAMPLE 10: Manipulations ofthe Environment that Increase Metal Availability A variety of different techniques can be used to increase metal availability in soils in accordance with the present invention. These tre~tment~ can be applied individually of separately.
Chelators As discussed above, many metal chelators act to increase metal mobility in soils(in addition to and distinct from any effect such chelators may have on inrlllcing metal transport into plant roots). For purposes of this section, an "effective amount" of a metal ch~l~tor is an amount sufficient to increase metal mobility but not sufficient to significantly alter plant growth and development. Desirable "effective amounts" of particular chelators are readily dPtermined through measurements metal mobility effects.
~or example, the concentration of soluble metals in soils can be measured according to the technique described by Mench et al. (J. Environ. Qual. 23:58, 1994, incorporated herein by reference). Briefly, metals are extracted from Sg of soil by equilibration with about 25 ml of 0.01 M calcium nitrate (to m~int~in ionic strength) for about 2 hours on a mechanical shaker. After the equilibration period, the suspension is CA 022493~3 1998-09-21 centrifuged (between 3000-5000 x g) for about 15 minutes to separate the solution from the soil. The supern~t~nt solution is then analyzed for the desired water-soluble metal concentration. Measured metal concentration is correlated with the amount and type of chelator added, so that optimal conditions for maximi7ing metal availability aredetermined.
Many metal chelators increase metal availability by forming soluble complexes with metals, thereby increasing metal solubility in the soil solutions. Exemplary solubilizing chelators include ammonium purpurate (murexide), 2,3-butane-dione dioxime (dimethylglyoxime), 3,6 disulfo-1,8-dihydroxyn~phth~lene (chromotroic acid), thiourea, alpha-benzoin oxime (cupron), trans-1,2-~i~rninocyclohexanetetraacetic acid (CDTA), diethylene-triaminopentaacetic acid (DTPA), 2,3-dimercapto-1-propanol, diphenylthiocall,azolle, nitrilotriacetic acid (NTA), substituted l,10-phPn~nthrolines (e.g., 5-nitro-1,10 phP,rl~nthroline), sodium diethyldithioc~balllate (cupral), 2-phenoyl-2-furoylmethane, phenoyl-trifluoroacetone, triethylenetetramine, EDTA, citric acid, EGTA, HEDTA, salicylic acid, and malic acid. (see Dawson et al., (eds) ,"Stability Constants of Metal Complexes", pp. 399-415, Data for Biochemical Research, Clarendon Press, Oxford, UK, 1986, incoll,oldled herein by reference).
Chelating agents are preferably applied to soil by conventional irrigation pipesor other ground level irrigation systems. Chelating agents may alternately be applied through commercially available fertilizer and chemical application equipment, including large ~lume sprayers. Chelating agents may be applied through broadcast methods for large areas or banding methods for the root zone. Chelating agents are preferably applied at concentrations from 0.1-10 mmol/kg soil.

CA 022493~3 1998-09-21 W O 97/34714 PCT~US97/~49S6 Acidification Also as discussed above, metal mobility in soil can be increased by decreasing the soil pH. Conventional methods of plant cultivation generally require soil in the pH
range 5.8-6.2 for optimum production and the available literature suggests that soils with lower pH be specifically amended with base (e.g., lime) prior to seeding to increase the pH (see, for example, "Agronomy of Canola in the United States", pp. 25-35 in Canola and Rapeseed, Production, Chemistry, Nutrition, and Processing Technology, ed. F. Shahidi, Van Nostrand Reinhold, New York, l 990, incorporated herein by reference).
In order to increase metal availability in the practice of the present invention, however, pH of the metal-co.-t~ ted is reduced to about pH 4.5-5.5 by acidifyingsoil with an effective amount of organic or inorganic acids (such as nitric acid, hydrochloric acid, sulfuric acid, acetic acid and citric acid). Acids are preferably applied to the soil by conventional irrigation pipes or other ground level irrigation systems. Acids may alternately be applied through other commercially available fertilizer and chemical application equipment, including large volume sprayers. Acids are preferably applied at concentrations from 0.1mM to 1.0 M at volumes ranging from about 5 to 200 tons per acre or at levels sufficient to drop soil pH in the plant rhizosphere (down to about 40 cm) to between 4.5 and 5.5 pH units.
Acidification of the plant environment may alternately be accomplished by addition to the environment of compounds that depress soil pH because of biological activity of roots and microor~ni~m~. Examples ofthese compounds include urea or ammonium sulfate. This so-called "biological acidification" occurs because the CA 022493~3 1998-09-21 positively charged ammonium ions that are incorporated into the roots and/or microorg~ni~m~ are replaced with positively charged protons exuded or otherwise released from the rhizosphere into the soil, thus lowering the soil pH. The ammonium-cont~ining compounds are applied at 0.5 to about 2.0 tons per acre.
Where acidification techniques are employed in combination with chelators, it isgenerally desirable to reduce the soil pH by at least 2 pH units over a period of several days, preferably to a pH within the range of about 3-4.5~ by adding strong chelators or acids prior to harvest but after the plants have reached the harvestable stage.

Electric fields Metal availability can be enhanced by using electrical fields to increase metal mobility (see, for example, Probstein et al., Science 260:498, 1993, incorporated herein by reference). In this method, a direct current electric field is applied across electrode pairs placed in the ground. The electric field induces motion of liquids and dissolved ions.

Soil Tilling Metal availability to plant roots can be increased by tilling soil to depths greater than 2 cm and as far down as 50 cm. Conventional implements may be employed for this purpose, provided that they are suitable for tilling down to the depths required by the present methods. These implements include moldboard plows, chisel plows, tandem and offset disc plows, and various harrowers known to those having ordinary skill in the art. The exact implement used will depend on factors understood in the art, such as soil CA 022493~3 1998-09-21 W O 97/34714 PCT~US97/04956 moisture, soil texture, weed cover and the like.

EXAMPLE 1 1: Inducing Hyperaccumulation of Uranium Materials and Methods:
SOIL CHARACTERIZATION: Uranium-cont~min~te~l soils were collected from an industrial site in northern Ohio. The soil was screened to pass through a 1.0 cm sieve, and was thoroughly mixed before use. The soil had a clay loam texture and an organic matter content of 4.1%. The soil pH (1:1 soil/water) was within the range of 5.5-7Ø
Uranium was present in the soil at 200-800 mg/kg (total); the solution concentration of uranium in the soil was 2-15 mg/L. The soil solution was extracted by a centrifugation method described by Elkhatib et al. (Soil Sci. Soc. Am. J. 51:578, 1987, incorporated herein by reference). Briefly, the soil was watered to field capacity and kept at room te~ .dl lre for 24 hr before the soil solution was extracted. The soil solution was passed through a 0.45 ~lm filter during the centrifugation, and the u~ l concentration in the soil was directly analyzed by ICP.
SOIL AMENDMENT APPLICATION: A variety of dirr~.ent soil ~men-lment~ were applied to the soil and those that most effectively i) solubilized the uranium (i.e., enh~nrecl desorption of the uranium from the soil into the soil solution); and ii) in~luçed uranium hyperaccumulation into plant shoots were identified. Specifically, selected plant species were grown on uranium-cont~min~tecl soils in a growth chamber for 4-6 weeks. Subsequently, selected soil amendments were applied to the root-zone of the plants. First, a stock solution was prepared for each soil ~men~ment (0.5 M for organic or inorganic acids; 0.1 M for sodium bicarbonate, potassium bicarbonate, or sodium CA 022493~3 1998-09-21 acetate). The ~p~ul~;ate amount of stock solution was then delivered to the root-zone of the plants. Plants were harvested one week after application of the soil amendments.
Plant samples were digested in a mixture of concentrated HNO3/HCl04, and the digested samples were analyzed for uranium content by ICP.

Results and Discussion Figure 7 shows the results of a study analyzing the effects of several differentsoil amendments on uranium availability in the soil. As shown, the solution concentration of uranium in the soil was increased approximately 2-200 fold, depending on the amendment applied. Citric acid was most effective at solubilizing the metal, increasing soil solution concentration from 1.2 mg/L to 240 mg/L. Addition of citric acid to the cont~min~ted soil transiently reduced soil pH by 0.5 to 1.0 pH unit. Because application of nitric acid had a similar effect on soil pH but a less dramatic effect on uranium solubility, we conclude that at least part of the solubilizing effect of citric acid is due to its ability to chelate the metal.
Figure 8 shows the effects of several acidifying agents on the induction of uranium hyperaccurnulation into B. juncea shoots. All of the agents in~ cecl at least about two-fold more accurnulation than occurs in the absence of any amendment. Citric acid was the most effective inducing agent, stimulating accumulation at least about 100-fold. We therefore applied citric acid to several individual B. juncea cultivars, and found that it was able to induce from 250 to 300-fold increases in u~aniul~ shoot accumulation (Figure 9). We also applied citric acid to a variety of different plant species and found that it was an effective inducing agent for all plants tested (Figure CA 022493~3 1998-09-21 10). Citric acid application increased metal accumulation at least about 2-fold in all species, and as much as about 1 OOO-fold in some.
Using the B. juncea cultivar (426308) that we found to accumulate the highest level of uranium after exposure to citric acid, we examined the time-dependent kinetics S of uranium accumulation. As shown in Figure 11, citric-acid-in(lllrecl hyperaccumulation was observed within 24 hours after amen~lment application. Theshoot uranium concentration reached a steady state three days after citric acid application.

EXAMPLE 12: Synergistic Induction of Uranium Hyperaccumulation by Exposure to both Altered pH and Herbicide Materials and Methods:
SOIL CHARACTERIZATION: Urar~ium-cont~min~t~d soils were collected and analyzed as described in Example 11.
SOIL AMENDMENT APPLICATION: A variety of different soil ~men-lments were applied to the soil and those that most effectively i) solubilized the uranium (i.e., enhanced desorption of the uranium from the soil into the soil solution); and ii) in(lllcecl uranium hyperaccumulation into plant shoots were identified. Specifically, selected plant species were grown on uranium-cont~min~ted soils in a growth chamber for 4-6 weeks. Subse~uently, both citric acid and foliar spray ROUNDUP~ solution (cont~ining 1.5% of concentrated ROUNDUP~) were applied to the plants. The plants were harvested one week after application of the soil arnendment. Plant samples were digested in a mixture of concentrated HNOJHC104, and the digested samples were analyzed for uranium content by ICP.

Results and Discussion:
As shown in Figure 12, when the citric acid level was less than 10 mmollkg, foliar spray ROUNDUP~ solution significantly increased the extent of uraniurn hyperaccumulation into plant shoots. These results indicate that, in the presence of foliar spray ROUNDUP~, significantly lower arnounts of citric acid are needed toachieve a specific level of uranium accumulation in plant shoots.

Other Embodiments The foregoing has set forth certain plef~ d embodiments of the present invention. The foregoing description is not meant to limit the scope of the present invention. One of oldin~y skill in the art will readily appreciate that various modifications and alterations are within the scope of the following claims.
What is claimed is:

Claims (49)

Claims

1. A method of inducing hyperaccumulation of a metal into shoots of a plant comprising;
planting a plant in a soil environment contaminated with one or more metals;
manipulating the soil environment to increase availability of metals in the environment to the plant;
cultivating the plant in the soil environment under conditions and for a time sufficient for the plant to accumulate metal in its roots; and exposing the plant to an inducing agent under conditions and for a time sufficient for the inducing agent to induce the plant to hyperaccumulate metal in its shoots.

2. The method of claim 1 further comprising a step of harvesting the plant shoots into which metal has been accumulated.

3. The method of claim 1 or 2 wherein the step of exposing comprises exposing the plant to an inducing agent under conditions and for a time sufficient that the plant accumulates more metal in its shoots than it would accumulate in the absence of the inducing agent.

4. The method of claim 3 wherein the step of exposing comprises exposing the plant to an inducing agent under conditions and for a time sufficient that the plant accumulates at least about twice as much metal in its shoots than it would accumulate in the absence of the inducing agent.

5. The method of claim 3 wherein the step of planting comprises planting a plant in a soil environment contaminated with one or more metals selected from the group consisting of actinium, aluminum, americium, antimony, arsenic, barium, beryllium, cadmium, cerium, cesium, chromium, cobalt, copper, curium, europium, gold, indium, iridium, lanthanum, lead, manganese, mercury, molybdenum, neodymium, nickel, palladium, rubidium, ruthenium, selenium, silver, strontium, technetium, thallium, thorium, tin, uranium, vanadium, yttrium, zinc, zirconium and combinations thereof with one another or with an organic contaminant.

6. The method of claim 5 wherein the step of planting comprises planting a plant in a soil environment contaminated with a metal that is not essential for plant growth.

7. The method of claim 5, wherein the step of planting comprises planting a plant in a soil environment contaminated with a metal selected from the group consisting of arsenic, cadmium, chromium, copper, lead, nickel, uranium and zinc.

8. The method of claim 3 wherein the step of planting comprises planting a plant having a characteristic selected from the group consisting of an ability to produce several crops per year, an ability to grow in various climates, an ability to grow in various soil conditions, an ability to grow when exposed to modified, non-conventional agricultural practices, an amenability to genetic manipulation, and an ability to grow to high biomass.

9. The method of claim 8 wherein the step of planting comprises planting a crop plant.

10. The method of claim 8 wherein the step of planting comprises planting a crop-related plant.

11. The method of claim 3 wherein the step of planting comprises planting a plant that is a member of the family Brassicaceae.

12. The method of claim 11 wherein the step of planting comprises planting a plant that is a member of a genus selected from the group consisting of Brassica, Thlaspi, Alyssum, and Eruca.

13. The method of claim 12 wherein the step of planting comprises planting a plant that is a member of a species selected from the group consisting of Brassica juncea, Brassica nigra, Brassica campestris, Brassica carinata, Brassica napus, and Brassica oleracea.

14. The method of claim 12 wherein the step of planting comprises planting a plant that is a Brassica juncea cultivar.

15. The method of claim 3 wherein the step of exposing comprises exposing the plant to an inducing agent selected from the group consisting of chelators, soil acidifiers, herbicides, detergents, heat, and radiation.

16. The method of claim 15 wherein the step of exposing comprises exposing the plant to a chelator selected from the group consisting of EDTA,EGTA,DTPA,CDTA, HEDTA,NTA, citric acid, salicylic acid, and malic acid.

17. The method of claim 16 wherein the step of exposing comprises exposing the plant to EDTA.

18. The method of claim 17 wherein the step of exposing comprises exposing the plant to a concentration of EDTA greater than about 0.2 mM.

19. The method of claim 15 wherein the step of exposing comprises exposing the plant to a soil acidifier selected from the group consisting of nitric acid, acetic acid, ammonium acetate, ammonium sulfate, ferrous sulfate, ferrous sulfide, elemental sulfur, sulfuric acid, citric acid, and ascorbic acid.

20. The method of claim 15 wherein the step of exposing comprises exposing the plant to reduced pH conditions by adding to the soil a metabolite that is processed by elements of the plant rhizosphere in a manner that produces protons.

21. The method of claim 19 wherein the step of exposing comprises exposing the plant to a soil pH to below about pH 5Ø

22. The method of claim 21 wherein the step of exposing comprises exposing the plant to a soil pH below about pH 3.5.

23. The method of claim 18 wherein the step of exposing comprises exposing the plant to an herbicide selected from the group consisting of glyphosate, MCPA, maleic hydrazide, 2,4-D, and combinations thereof.

24. The method of claim 15 wherein the step of exposing comprises exposing the plant to a combination of chelating agent and soil acidifier.

25. The method of claim 24 wherein the chelating agent is selected from the group consisting of EDTA, EGTA, DTPA, CDTA, HEDTA, NTA, citric acid, salicylic acid, and malic acid, and the soil acidifier is selected from the group consisting of nitric acid, acetic acid, ammonium acetate, ammonium sulfate, ferrous sulfate, ferrous sulfide, elemental sulfur, sulfuric acid, citric acid, ascorbic acid, and metabolites that are processed by elements of the plant rhizosphere in a manner that produces protons.

26. The method of claim 15 wherein the step of exposing comprises exposing the plant to a combination of chelating agent and herbicide.

27. The method of claim 26 wherein the chelating agent is selected from the group consisting of EDTA, EGTA, DTPA, CDTA, HEDTA, NTA, citric acid, salicylic acid, and malic acid, and the herbicide is selected from the group consisting of glyphosate, MCPA, maleic hydrazide, 2,4-D, and combinations thereof.

28. The method of claim 27 wherein the step of exposing comprises:
exposing the plant to the chelating agent;
waiting a period of time; and exposing the plant to the herbicide.

29. The method of claim 15 wherein the step of exposing comprises exposing the plant to an acidifying agent and an herbicide.

30. The method of claim 29 wherein the acidifying agent is selected from the group consisting of nitric acid, acetic acid, ammonium acetate, ammonium sulfate, ferrous sulfate, ferrous sulfide, elemental sulfur, sulfuric acid, citric acid, ascorbic acid, and metabolites that are processed by elements of the plant rhizosphere in a manner that produces protons and the herbicide is selected from the group consisting of glyphosate, MCPA, maleic hydrazide, 2,4-D, and combinations thereof.

31. The method of claim 30 wherein the step of exposing comprises:
exposing the plant to the acidifying agent;
waiting a period of time; and exposing the plant to the herbicide.

32. The method of claim 1 wherein the step of manipulating comprises applying an effective amount of a chelating agent.

33. The method of claim 32 wherein the chelating agent is selected from the group consisting of murexide, dimethylglyoxime, chromotroic acid, thiourea, cupron, CDTA, DTPA, NTA, substituted 1,110-phenanthrolines, cupral, 2-phenoyl-2-furoylmethane, phenoyl-trifluoroacetone, triethylamine, EDTA, citric acid, EGTA, HEDTA, salicylic acid, and malic acid.

34. The method of claim 1 wherein the step of manipulating comprises reducing the soil pH to about pH 3.0-5.5.

35. The method of claim 34 wherein the step of manipulating comprises reducing soil pH through application of an effective amount of an organic or inorganic acid selected from the group consisting of nitric acid, hydrochloric acid, sulfuric acid, acetic acid, malic acid and citric acid.

36. The method of claim 34 wherein the step of manipulating comprises reducing soil pH through application of a compound that is metabolized by the plant rhizosphere in a manner that produces protons.

37. The method of claim 1 wherein the step of manipulating comprises applying an electric field to increase metal mobility.

38. The method of claim 1 wherein the step of manipulating comprises tilling the soil to a depth greater than about 2 cm before plants are planted therein.

39. A method of removing metal from an environment contaminated with the metal, the method comprising:
planting a plant that is a member of the family Brassicaceae in the environment;
applying an agent selected from the group consisting of chelating agents, acidifiers, and combinations thereof to the environment to increase metal availability to the plant planted therein;
waiting for a period; and applying an herbicide to the environment to induce hyperaccumulation of metal in shoots of the plant.

40. The method of claim 39 wherein the plant is a member of the genus selected from the group consisting of Brassica, Thlaspi, Alyssum, and Eruca.

41. The method of claim 40 wherein the plant is a member of a species selected from the group consisting of Brassica juncea, Brassica nigra, Brassica campestris, Brassica carinatam Brassica napus, and Brassica oleracea.

42. The method of claim 41 wherein the plant is a Brassica juncea cultivar.

43. In a method of removing metal from an environment by cultivating a plant therein, the improvement that comprises:
exposing the plant to an inducing agent under conditions and for a time sufficient to induce the plant to hyperaccumulate metal in its shoots to a level higher than the plant would accumulate in the absence of the inducing agent.

44. The method of claim 43 wherein the inducing agent is selected from the group consisting of chelating agents, soil acidifiers, and herbicides.

45. The method of claim 43 wherein the plant is a Brassica juncea cultivar, the metal is lead, and the inducing agent is selected from the group consisting of at least 0.2 mM EDTA, pH less than about 3.5, and an herbicide selected from the group consisting of glyphosate, 2,4-D, and combinations thereof.

46. The method of claim 43 wherein the metal is selected from the group consisting of cadmium, copper, nickel, lead, and zinc.

47. The method of claim 43 wherein the metal is uranium.

48. The method of claim 60 or claim 47 wherein the inducing agent is selected from the group consisting of a chelator, an herbicide, and combinations thereof.

49. A method of identifying an agent that induces hyperaccumulation of a metal into plant shoots, comprising steps of:
cultivating a plant in an environment contaminated with a metal;
exposing the plant to a potential inducing agent; and identifying an agent that induced the plant to accumulate higher levels of metal in its shoots when exposed to the agent than when not so exposed.