Remediation of Potentially Toxic Elements Through Transgenic Plants

C H A P T E R

6 Remediation of Potentially Toxic Elements Through Transgenic Plants: In Vitro Studies and the Way Forward Mohamed Cassim Mohamed Iqbal1 and Sithy Sabeera Iqbal2 1

Plant and Environmental Sciences, National Institute of Fundamental Studies, Kandy, Sri Lanka, 2 Department of Chemistry, The Open University of Sri Lanka, Nawala, Sri Lanka

6.1 INTRODUCTION Using green plants to remove pollutants from our environment is a very old technology. An early reference to this was the observation, by the German botanist A. Baumann in 1885, that plant species growing in soil naturally enriched in Zn accumulated high levels of this element in their leaves (Salt et al., 1998). Since then many species have being identified capable of absorbing different heavy metals from the soil environment.

6.1.1 Toxic Metals and Metalloids in the Environment Emission of toxic heavy metals and metalloids has risen tremendously and significantly exceeds those from natural sources for practically all metals (Prasad, 2004; Clemens, 2006). Of the numerous anthropogenic activities,

Transgenic Plant Technology for Remediation of Toxic Metals and Metalloids DOI: https://doi.org/10.1016/B978-0-12-814389-6.00006-7

mining operations are potential sources of airborne metal and metalloid contaminants through both direct smelter emissions and wind erosion of mine tailings (Csavina et al., 2012) where fine particulates from smelting operations may disperse more readily into the environment than coarser tailings dust. Contaminants can be transported rapidly and over relatively long distances by atmospheric dust and aerosol relative to other media such as water, soil, and biota (Csavina et al., 2012). The impact of abandoned mine wastes on the levels of metals and metalloids in the atmosphere decreases with increasing distance from the mine waste deposits (Castillo et al., 2013). Large-scale Pb/Zn alloy smelters have also contributed to elevated levels of trace elements in street dust samples as reported from a heavily industrialized city in central China (Li et al., 2013). Such smelt also causes heavy metals (Hg, Pb, Zn, Cd) and metalloid (As)

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pollution of nearby soil (Li et al., 2011). Uncontrolled electronic waste recycling operations have also caused serious heavy metal pollution to local soils and vegetation (Luo et al., 2011). In all other cases, vehicle-related sources are found to be important (Manalis et al., 2005). Heavy metals and metalloids in soils are derived from their parent material and various anthropogenic sources (Alloway, 2013). Although the heavy metal(loid) composition of agricultural soils is closer to that of the parent material, deposition of aerosol particles transported long distance in the atmosphere from fossil fuel combustion facilities and industrial sites can significantly alter it. In general, soils at industrial sites can have distinct groups of heavy metal(loid) contaminants, which depend on the respective industries and their raw materials and products. Soils in all urban areas are generally contaminated with lead (Pb), zinc (Zn), cadmium (Cd), and copper (Cu) from traffic, paint, and many other nonspecific urban sources. Of the many different anthropogenic sources of heavy metal(loid)s that contaminate the soils, a metal smelter can have a marked effect on the quality of soils. Though strict regulations on atmospheric emissions and wastewater discharges have resulted in a general reduction in the loads of heavy metal(loid)s reaching soils in many countries, historic contamination still affects soils in many areas and may have long term impacts (Alloway, 2013). Often mining of metals can lead to release of other toxic metals and metalloids as reported in the case of the abandoned mercury mine in Spain where high levels of arsenic were found in the terrestrial and aquatic environments arising from the arsenic-rich minerals associated with the mercury ore (Loredo et al., 2005, 2006).

6.1.2 Phytoremediation of Metals and Metalloids Unlike organic contaminants, metals and metalloids do not undergo microbial or

chemical degradation and persist for a long time after they are released into the environment (Bolan et al., 2014). Therefore, there is an urgent need to develop low-cost, effective, and sustainable methods to remove them from the environment or detoxify them. Plant-based approaches, such as phytoremediation, are relatively inexpensive since they are performed in situ and are solar-driven (LeDuc and Terry, 2005). Advances in these approaches to remediate contaminated water and soil include use of constructed wetlands to remove trace elements in dilute concentrations from large volumes of wastewater and use of genetic engineering to develop plants with enhanced efficiency to accumulate metals/metalloids in their harvestable biomass (phytoextraction) and convert and release certain metals/metalloids in a volatile form (phytovolatilization) (LeDuc and Terry, 2005; Prasad, 2013). Bioavailability of metals and metalloids plays an important role in the remediation of contaminated soils (Bolan et al., 2014). Bioavailability of metal(loid)s, can be increased or decreased using a range of soil amendments to mobilize or immobilize metal(loid)s, respectively, to remediate soils contaminated by them. Chelating and desorbing agents increase the bioavailability and mobility of metal(loid)s and enhance removal of the metal(loid)s through plant uptake (phytoextraction) while precipitating agents and sorbent materials decrease the bioavailabilty and mobility of metal(loid)s, thus immobilizing them (Bolan et al., 2014). In the study of heavy metal(loid) pollution in aquatic systems, investigation into the level of metal(loid)s in surface water and sediments has been carried out simultaneously for a meaningful conclusion about the pollution status. In a study of heavy metal pollution in surface water and sediments in Bangladesh, levels of Cr, Ni, Cu, As, Cd, and Pb were found to exceed the safe limits of drinking water while contamination factor (CF) and geoaccumulation index (Igeo) demonstrated that most of the

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sediment samples were moderately to heavily contaminated by Cr, As, Cd, and Pb (Islam et al., 2015). Mining waste from extraction of gold and silver from ores in Romania have introduced heavy metals and metalloids, such as Pb, Zn, Cd, Cu, Ni, and As, in agricultural land and surface and ground water in concentrations exceeding the pollution threshold (Modoi et al., 2014). Analysis of sediments and water of the Seyhan River in Turkey showed very low levels of heavy metals while the sediments of the river demonstrated “unpolluted to moderate pollution” due to Cu, Pb, and Zn and “moderate to very strong pollution” due to Cr and Ni according to sediment quality guidelines (SQG) of the US-EPA (Davutluoglu et al., 2011). The study of heavy metal pollution in water and sediments in the river Kabini in Karnataka, India found, as expected, higher concentrations of metals in the vicinity of discharge of industrial effluents (Hejabi et al., 2011). In this context, plants offer a viable alternative to remove pollutants. They are a selfcontained system: they are autotrophic utilizing solar energy to synthesize their food using carbon, nitrogen, potassium, and other minerals from the environment. Besides heavy metals, plants are also capable of absorbing and enzymatic breakdown of foreign organic compounds such as pesticides (Sandermann, 1994), chlorinated solvents, polyaromatic hydrocarbons, and explosives (Pilon-Smits, 2005; Salt et al., 1998). However, this capability is limited since plants are autotrophic and are not dependent on organic compounds as a source of energy or for carbon metabolism (Van Aken, 2008). Plants have different strategies to tolerate and remove pollutants from soil and water, which go under the generic term phytoremediation. This term includes phytoextraction, the most common form of phytoremediation. The contaminant metals are absorbed through the roots, and translocated to the shoots, where they are sequestered, and the contaminant is

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removed from the environment by simply removing the plants. Rhizofiltration is the removal of pollutants from aquatic environments. In plant tissue culture the phenomenon of hairy roots is induced by infecting explants with the soil bacterium Agrobacterium rhizogenes. Hairy roots have been successfully used to remove organic and inorganic pollutants, reviewed by Agostini et al. (2013). Certain species such as Thlaspi caerulescens (Nedelkoska and Doran, 2000), Brassica juncea, and Chenopodium amaranticolor (Eapen et al., 2003) were particularly effective in removing radionuclides and heavy metals. Certain heavy metals such as Hg, Se, and arsenate are suitable for volatilization through the plant. Although volatilization of Hg and As is possible by microorganisms, plants cannot significantly volatilize these pollutants (Cherian and Oliveira, 2005). The efficiency of phytoremediation can be enhanced by identifying and selecting appropriate species or varieties and using agronomic practices to enhance the mobility of pollutants such as heavy metals into the plant (PilonSmits and Pilon, 2002). These include increasing the plant biomass through fertilizer application and adding soil amendments to increase the transport of heavy metals such as chelating agents or organic acids (Chaney et al., 2000; Blaylock and Huang, 2000). Plant scientists have two options to enhance phytoremediation. Species and varieties can be screened and selected for uptake of a particular pollutant. This can be extended to breeding by crossing varieties or selections within a species to increase the frequency of desirable genes such as a high rate of pollutant uptake, high biomass, tolerance to pollutants, survival ability in a polluted environment, etc. Conventional breeding, however, could take many generations. A more rapid approach is to introduce a foreign gene that is known to contribute to the remediation of a pollutant. This could be through uptake, degradation

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after uptake, or sequestration of the pollutant (Kotrba et al., 2009; Pavlikova et al., 2004). The recent past has seen a plethora of reviews on the potential for transgenics in phytoremediation for a variety of environmental pollutants. These include transgenic approaches to remediate organic and inorganic pollutants and the use of trees (Cherian and Oliveira, 2005), the remediation of recalcitrant organic xenobiotics (Eapen et al., 2007; Abhilash et al., 2009), an overview of genes associated with hyperaccumulation and tolerance to heavy metals (Sarma, 2011), transgenics and heavy metal tolerance in edible and medicinal plants (Fahimirad and Hatami, 2017), the potential for transgenics for phytoremediation of heavy metals and metalloids (Fasani et al., 2018), the application of plant glutathione transferases in phytoremediation (Nianiou-Obeidat et al., 2017), among others. Book chapters in Bioremediation of Aquatic and Terrestrial Ecosystems provide a comprehensive review across many themes including genetic engineering of microorganisms for bioremediation and strategies to remediate wetlands, oil spills, and explosives, among others (Fingerman and Nagabhushanam, 2016). This chapter attempts to highlight the recent advances in transgenic research to remediate heavy metals from the environment and the role of in vitro studies. The challenges faced in the introduction of transgenics and the means to overcome these are discussed.

6.2 TRANSGENICS IN PHYTOREMEDIATION 6.2.1 Source of Transgenes Microorganisms, particularly bacteria, are single celled and in the absence of a robust cell wall are more susceptible to environmental pollutants. They have been exposed to heavy metals in the environment since the beginning of life on Earth. By virtue of their short generation interval, they

have accumulated genes capable of metabolizing pollutants and resisting toxic concentrations of almost all the toxic metals such as Ag1, AsO22, 21 21 21 21 21 22 AsO32 4 , Cd , Co , CrO4 , Cu , Hg , Ni , 21 l1 21 22 Pb , TeO3 , T , and Zn (Silver and Phung, 2005). However, they cannot be readily used for bioremediation. Since they are heterotrophic, they need a source of nutrients. They also need to be cultured and inoculated under conditions that permit their growth besides the necessity to harvest them if the pollutants are not degraded completely. Besides, their performance under different environments in the laboratory and under contaminated field conditions is not always similar (Goldstein et al., 1985). The genes that the bacteria have evolved, however, can be exploited by genetically engineering them into suitable higher plants and thus are a good source of genes for transformation. The resistance systems in microorganisms function mostly by energy-dependent efflux of the toxic ions and others by enzymatic transformations such as oxidation, reduction, methylation, and demethylation or metal-binding proteins such as metallothioneins, among others (Silver and Phung, 2005). They can also use organic pollutants—xenobiotics—as a carbon source. Although microorganisms have a larger surface area to absorb metals, plants have the advantage of deeper penetrating roots in the soil to access heavy metals that are in the root zone. Plants that have colonized physically or chemically harsh environments are also a source of genes for phytoremediation. A good source of metal hyperaccumulating plant species is soil naturally rich in heavy metals such as those in serpentine sites (Harrison and Rajakaruna, 2011; Galey et al., 2017) and regions where heavy metals are mined and abandoned. Serpentine soils are not only rich in heavy metals such as Ni, Fe, and Mn and the macronutrient Mg but also are a harsh environment with very poor soil structure, organic matter, and macronutrients. Thus, species growing on these soils have

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adapted to tolerate high levels of heavy metals in their tissues (Kazakou et al., 2008) and also tolerate unfavorable environments—characteristics to be expected in sites for phytoremediation. Such species are most likely the result of mutations in their genome and natural selection and are a good source of phytoremediators and genes for cloning.

6.2.2 Plant Characteristics Desired for Phytoremediation Plants are autotrophic and are easily propagated through seeds; they need only sunlight, water, and a source of inorganic ions usually available in the soil. Plants need to meet certain conditions to be used as phytoremediators: ideally, they should not be edible, should accumulate biomass rapidly, and show robust growth under harsh environments. Plants are thus an environmentally benign means of remediating a contaminated site. The desired plant attributes for phytoremediation fall into two broad categories: morphological and physiological. 6.2.2.1 Morphological Attributes At the macro level of the plant, the following attributes are highly desirable, although they may not all occur in the same species: • rapid attainment of high biomass; • tolerate broad environmental characteristics (climate, soil, water); • widespread root system—in dicots, deep tap root and horizontal spread; in monocots, dense spread of roots on the surface soil; • nondeciduous; • good root to shoot translocation of the metal pollutant; • propagation by vegetative means. These plant characteristics are under polygenic control and their improvement by conventional breeding with selection can take 5 6

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years, depending on the generation interval of the species. Tree species are perhaps better as phytoremediators. They have a high biomass, are deep rooted, and if tolerant to heavy metals are ideal for phytoremediation. They can also be a source of dendropower in bioenergy industries if the timber is hardwood for construction and manufacturing purposes (Rockwood et al., 2004). Trees are well adapted to grow on marginal soils, which is what remains after extensive mining operations to extract metals from the earth. The major advantages of trees in phytoremediation are: • perennial growth and accumulation with no need for annual harvest; • well-developed root system in mature trees; • effective water uptake, which can be used to stabilize the polluted site; • production of litter that provides nutrients to poor soils after decomposition; • some potential tree species for phytoremediation are also of economic importance. One such species is Salix matsudana, which is native to northeast China and is capable of accumulating high levels of heavy metals (Utmazian et al., 2007). However, this species takes a long time for extracting the heavy metals and the translocation from roots to shoots is low, which is undesirable for harvesting the aboveground biomass with the heavy metals (Dowling and Doty, 2009). To overcome these restrictions, Yang et al. (2015) transformed S. matsudana with a type 3 metallothionein gene isolated from Tamarix hispida. This gene, ThMT3, not only increased the tolerance to Cu in the transgenic plants but also induced numerous adventitious roots under Cu stress. The application of a nitrous oxide inhibitor to the transgenic plants under Cu stress decreased the activity of superoxide dismutase, catalase, and ascorbate peroxidase. The authors further

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found that genes related to auxin application, which improve adventitious roots, were highly expressed under Cu stress in the transgenic plants. Thus, the overexpression of the ThMT3 gene in S. matsudana increased tolerance to Cu and rooting efficiency of the transgenic plants and the NO levels were significantly higher than in the wild type plants. 6.2.2.2 Physiological Attributes The second characteristic to be targeted is the physiological process of absorbing the metal pollutants and sequestering them within the plant. The genetic engineering of plants for phytoremediation is based on the ability of plants to tolerate the metals. The basic strategies by the cell to achieve this is by either keeping the concentrations of the toxic metals low within the cell through exclusion or by detoxifying the metal ions in the cytoplasm through chelation, conversion, or sequestration (Tong et al., 2004). Plant species differ in their ability to tolerate heavy metals. This depends on the uptake, root to shoot translocation, and their sequestration in vacuoles within the cells. The molecular mechanisms involved in these physiological processes can be the targets for transformation. Once the physiological process that needs to be introduced or enhanced is identified, they are amenable to genetic engineering. Processes that have been genetically modified include (Pilon-Smits and Pilon, 2002): • proteins involved in the transport of metals across cell membranes; • molecules capable of chelating and facilitating the transport of the metal; • proteins/enzymes involved in modifying the metals; • processes necessary to sequester the metals within the tissues; • processes that enhance the rhizosphere environment for uptake of metals by excreting chelates and organic acids to alter pH.

It is necessary to identify the biochemical pathways to understand the genes and enzymes involved in the phytoremediation process, so as to identify the pathways to be targeted for genetic transformation. This would enable the tailoring of specific gene/s to be introduced into the target plant. Heavy metals are ferried into plant cells by membrane transporter proteins. The primary role of transporter proteins is to absorb plant nutrients through the root system. However, heavy metals that are structurally and chemically similar to the nutrients also inadvertently enter the plants. The subsequent translocation of these heavy metals from the roots to the shoots is facilitated by a group of peptides called phytochelatins and metallothioneins. The heavy metals are then subsequently isolated in vacuoles so as not to interfere with other metabolic processes in the cell. Plants are totally dependent on the soil environment for their source of major or macronutrients (N, P, K. Ca, Mg, and S) and the minor or micronutrients (B, Cl, Cu, Fe, Mo, Mn, Ni, and Zn). Although the availability of these elements is varied in the soil, plants have evolved cellular mechanisms to maintain their correct concentrations within the cell—the phenomenon referred to as cellular homeostasis. Vacuoles, which in mature cells can take up 80% of the cell volume, are an important location where minerals and metabolites required by the cell are stored and are also used to isolate toxic compounds and heavy metals to prevent interference of cellular metabolism. Peng and Gong (2014) have suggested that the sequestration capacity of the vacuoles could dynamically mediate the long-distance transport of metals from roots to shoots. In plant metal hyperaccumulators, the excess metals absorbed into the roots are subject to long distance transport from roots to shoots where there is an enhanced capacity in the vacuoles to sequester the toxic metals, which is facilitated by metal chelators in the cytosol and transporters in the tonoplast (Gong et al., 2003; Peng and Gong, 2014).

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6.2.3 Genes for Phytoremediation

metal homeostasis and detoxification (Blindauer and Leszczyszyn, 2010). This is supported by evidence that correlates their discrete spatial and temporal expression profiles with specific types of plant metallothioneins (Leszczyszyn et al., 2013). The overexpression of plant metallothioneins under stress of the heavy metals Cu, Zn, and Cd led to resistance against these metals (Cobbett and Goldsbrough, 2002). After insertion of the metallothioneins gene into a suitable host plant its detection alone, however, is insufficient to confirm the phytoremediation ability; it is also necessary to determine the metallothioneins content in the transgenic plant (Diopan et al., 2008).

A number of genes are available in microorganisms and a few in plants for their potential use to enhance phytoremediation. These genes code for peptides that play a role in regulating the heavy metals in the cellular homeostasis of plants. The major groups of these proteins and peptides are metallothioneins, glutathiones, phytochelatins, and membrane transporters, which are known to regulate the levels of free metal ions in plant tissues (Cobbett and Goldsbrough, 2002). These are briefly discussed below and schematically illustrated in Fig. 6.1. 6.2.3.1 Metallothioneins Metallothioneins are low molecular weight cysteine rich metal-binding proteins (,10,000 Da) present in all eukaryotes and some prokaryotes (Leszczyszyn et al., 2013; Yang et al., 2015). Their structure enables them to bind to mono- and divalent ions, particularly Cu(I), Zn (II), and Cd(II) and also contribute to cellular

6.2.3.2 Plant Metal Chelators Plant metal chelators include nicotineamines, glutathione, and phytochelatins, which also facilitate long distance transport of metals from roots to shoots (Peng and Gong, 2014). Phytochelatins are thiol rich peptides, which

Functions

Peptides

Detoxifying ROS Metallothioneins

Homeostasis

Plasma lemma Transport out of the cell in to cell wall

T

Chelators Detoxify ROS Glutathiones Phytochelatines

Combine with metals Transport of metals

Cytoplasm

Vacuole

Transport in to vacuoles

Cellulose cell wall

T

Tonoplast

FIGURE 6.1 Physiological processes for metal tolerance, detoxification, and sequestration in plants: targets for transformation. ROS, Reactive oxygen species.

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can bind to heavy metal ions to form a nontoxic heavy metal protein complex thereby decreasing their toxicity within the cell. The phytochelatin metal complex can be further isolated in the plant cell vacuoles (Yadav, 2010). Phytochelatins are not encoded in the DNA— they are produced from glutathione by phytochelatin synthase, which is regarded as an important enzyme in phytoremediation (Cobbett and Goldsbrough, 2002). Phytochelatin synthase genes have been cloned from different species and their role in heavy metal resistance and accumulation is confirmed. Glutathione is an important molecule in plant cells for ionic homeostasis and for tolerating heavy metals (Helbig et al., 2008). It is a precursor of phytochelatins necessary for metal sequestration in cell vacuoles (PilonSmits, 2005). The molecule is highly water soluble and stable (Rausch et al., 2007). Thus, it is an ideal molecule to integrate into the genome of a potential metal hyperaccumulator species. 6.2.3.3 Membrane Transporters The transport of heavy metals across membranes are regulated by transporters (NRAMP3, NRAMP4) and chelators (phytochelatins, nicotine-amines). The ABC (ATPbinding cassette) transporters are one of the families of transporters well characterized in the vacuolar sequestration of heavy metals (Song et al., 2003). These major classes of proteins and peptides, among others, are the major targets for gene cloning and transformation of plants for phytoremediation. For effective phytoextraction, the transfer of the absorbed heavy metals from the roots to the shoots is important. Overexpression of ABC transporters significantly enhanced the ability to tolerate and accumulate heavy metals in the aerial parts (Li et al., 1997; Song et al., 2003; Kim et al., 2006, 2007). This has been studied in different species: the Nramp transporter in A. thaliana for Fe (Curie et al., 2000), CAX2 antiporter in N. tabacum (Hirschi et al., 2000), Zn

transporter in A. thaliana (Kobae et al., 2004), Zn and Cd transport from roots to shoots in A. thaliana (Verret et al., 2004), Zn and Cd accumulation in B. juncea (Xu et al., 2009), among others. Another gene in the ABC transporter family is AtATM3 found in the mitochondrial membrane of A. thaliana. Bhuiyan et al. (2011) transformed B. juncea with the YCF1 gene to improve heavy metal tolerance and accumulation. The transgenic plants had 1.5- to 2.5-fold higher Cd and Pb levels than the control wild type seedlings. The authors attributed this to the increased expression of glutathione synthetase and phytochelatin synthase brought about by the overexpression of AtATM3.

6.3 THE ROLE OF IN VITRO STUDIES 6.3.1 Screening for Efficient Uptake The first experimental system at the laboratory level is based on in vitro methods. These are necessary to screen and evaluate the efficiency of metal uptake, translocation, and sequestration in a transgenic experiment from the cell cultures of microorganisms to shoot cultures of perennial trees. The defined nutrient medium and the controlled manipulation of the variables provide a platform to gather data from sufficient replicates to test hypotheses on the efficacy of the transformations. In vitro experiments have a central role in determining the gene to be cloned from a microorganism or a plant, the process of genetic transformation, and establishing the successful transformation. Before attempting transformation, the identified gene should be cloned, and the mechanism of how the gene functions should be established. The source of genes to enhance phytoremediation are from microorganisms or hyperaccumulating plant species. Following transformation, the immediate testing of the transformed plants needs to

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be conducted under sterile, controlled, and reproducible conditions to provide information on mechanisms of uptake, translocation, and cellular sequestration. This stage of in vitro studies can confirm the integration of the desired gene in the genome of the desired plant species. However, the ultimate evaluation of the remediation potential needs to be confirmed under field conditions. In vitro systems provide a controlled means of evaluating this complex interaction to study the effect of stress factors on metabolism, specific enzymes, and metabolites involved in the plant response to the pollutants (Golan-Goldhirsh et al., 2004). Trees are perennial and in vitro culture is a useful tool to investigate their potential for phytoremediation. Capuana (2011) has briefly reviewed a number of species—Eucalyptus, Populus, Ailanthus, Acer, Pinus, and Betula— that were investigated using in vitro cultures for their phytoremediation potential of heavy metals. Unlike field based experiments at the early stages of screening, in vitro systems can generate a large volume of data under controlled conditions. This permits the evaluation of one or a few variables, and particularly independent of soil and climate factors, which can confound the primary effects under study. Di Lonardo et al. (2011) conducted an in vitro evaluation of three clones of Populus alba using their microshoots. One fast growing clone, although it did not have the highest metal content, showed that biomass production was a key factor in evaluating the phytoextraction capacity. Golan-Goldhirsh et al. (2004) have reviewed a wide spectrum of phytoremediation experiments of xenobiotics and heavy metals from in vitro cultures to whole plant level. They conclude that while in vitro methods provide explanations to the mechanisms of detoxification, uptake of contaminants and their metabolism, they do not predict the practical potential of these plants and enzyme systems in a straightforward manner. The authors suggest

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that a comprehensive approach be undertaken with multidisciplinary specialists—cell biologists, biochemists, agronomists—to undertake eventual field performance to solve problems of soil contamination. The three factors necessary for successful phytoremediation—heavy metal tolerance, uptake, and translocation from roots to shoots—is difficult to test in perennial trees. In vitro testing offers a solution provided the species can be propagated by in vitro methods. They also require less space and a shorter time duration to complete the testing process (Di Lonardo et al., 2011). The potential for phytoremediation of perennial and/or vegetatively propagated species can be investigated using in vitro systems, if protocols are available for in vitro multiplication through micropropagation. In vitro testing should be ideally followed by testing in a hydroponics system whose results show better agreement with field testing (Pulford and Watson, 2003; Watson et al., 2003).

6.3.2 From the Laboratory to the Field The path from successfully transforming a plant species, to using the plant for soil decontamination, faces many challenges. Many species have been transformed with a range of genetic mechanisms to enhance the uptake of heavy metals from the environment, their translocation within the plant, and their sequestration (Fasani et al., 2018). These studies have been tested using in vitro studies and hydroponic systems. The few studies that were field tested have provided mixed results indicating that the incorporation of the genes alone into a plant, its expression, and in vitro uptake do not guarantee its potential for remediation. After the first steps of cloning the gene and transforming the plant, the initial screening experiments are conducted under controlled laboratory conditions to obtain reproducible

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results. These are either under sterile in vitro conditions on agar solidified medium or hydroponic conditions, where the nutrients and heavy metals given to the plant are carefully formulated. In vitro culture environments are characterized by low irradiance and high humidity whereas the natural environment has a high and variable irradiance and relative humidity. Tissue cultured plants in vitro have a poorly developed cuticle on the leaves, and their photosynthetic activity is much reduced compared with plants under sunlight due to reduced levels of photosynthetic pigments. They need to be gradually hardened before release into the environment. Using a hydroponics system overcomes these limitations of in vitro studies. However, testing terrestrial species using hydroponics can introduce physiological aberrations to the uptake and exclusion of ions (Tavakkoli et al., 2012). Besides, the metals in the solution are easily bioavailable (Fasani et al., 2018) whereas in the soil they are found in different chemical forms and oxidation states or complexes with organic components (see Peijnenburg and Jager, 2003). In in vitro and hydroponics culture, the root system is completely deprived of oxygen, which can create changes in the absorption of ions by the roots. In experiments to screen for salinity tolerance, Tavakkoli et al. (2012) found that roots of barley partially excluded Na ions from shoots over a wide range of NaCl concentrations from 0.2 to 200 mM. This exclusion broke down under root anoxia (hydroponic culture) so that more Na ions reached the shoots with a simultaneous inhibition of K ion transport. Besides, the concentration of salts (and heavy metals) in the soil solution can change due to mass flow exceeding uptake and further due to decreasing water content from transpirational demand of the plant (Tavakkoli et al., 2012). In vitro experiments are limited spatially and temporally, and conducted under controlled

conditions with few variables to understand and collect evidence on the mechanism of uptake, translocation, and sequestration of the pollutant. Golan-Goldhirsh et al. (2004) have pointed out the gap between the rapid accumulation of data from in vitro experiments (cell cultures, hydroponics), which are small scale and whole plant in situ experiments under field conditions. When the in vitro level observations are not realized under field conditions, the transgenic system should be redesigned (e.g., gene stacking) to move towards the ultimate goal of in situ phytoremediation. Field testing of transgenic plants should meet stringent regulatory conditions at the national level. Thus, it is worthwhile to first conduct reliable trials under contained greenhouse conditions. To simulate field conditions, it would be appropriate to use soil from a contaminated site where remediation with the transgenic plants is foreseen as large pot experiments or in cement tanks. While these simulated conditions would overcome the major concerns of in vitro studies and hydroponic systems the simulation of the natural water flow in the soil would remain a challenge to overcome.

6.3.3 Evaluation of Transgenics After transformation of the plant species with the gene/genes to enhance phytoremediation, the transformed plant is first evaluated under in vitro conditions. Some of the studies have progressed to hydroponics systems while a few were also assessed in pot experiments with soil. A selection of these experiments is summarized in Table 6.1 (experiments with only in vitro testing), and Table 6.2 (experiments with hydroponics and soil testing) where different gene systems were used to transform, and the source of genes were mostly bacteria, with a few from plants. Some selected studies are discussed below.

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TABLE 6.1 Genes Introduced to Enhance Phytoremediation Capacity in Selected In Vitro Experiments and Their Performance (TR Transgenic, WT Wild Type) Product and Heavy Metal Metal transporter

Gene Transferred and Source PaMT Populus alba

Transformed Plant P. alba cv. Villafranca

In Vitro Test

Results and Comments

Aseptic shoot cultures on /2 Woody Plant Medium 1 phytagel. Treated with 0.5 to 4 mM ZnSO4. Toxicity (chlorosis) and rooting were evaluated

Castiglione Gene expression of 2 TR et al. (2007) clones were increased by Zn. TR clone was tolerant to external Zn concentrations of 5 , 1 mM. Metal taken up was translocated to shoots

1

Zn

Reference

Positive and dose dependent relationship between Zn and metallothionein transcript levels Metal transporter

YCF1

A. thaliana

TR plants were germinated on 1/2 MS.

S. cerevisiae

Pb and Cd

ABC transporter Cd, Pb

Plants overexpressing YCF1 have enhanced tolerance to Pb(II) and Cd(II)

Song et al. (2010)

YCF1 is an efficient mechanism to transport Pb and Cd into the vacuoles and enable detoxification AtATM3 A. thaliana

B. juncea Agrobacterium mediated transformation

WT and TR seedlings on /2 MS agar with 0.15 M CdCl2 and 1 M Pb(NO3)2. Analysis after 7 days 1

Enhanced tolerance to Cd (1.3- to 1.6-fold), Pb (1.2- to 1.4-fold) per g/FW

Bhuiyan et al. (2011)

Shoot tissues had 1.5 to 2fold higher Cd and Pb than WT Higher glutathione synthetase II and phytochelatins synthase expression induced by AtATM3 Glutathione transferases Cd and trichloroethylene (TCE)

pKHCG GST and CYP2E1

Medicago sativa

Sterile seeds of TR and WT were grown on MS medium with different concentrations of CdSO4 and trichloroethylene for 20 days to evaluate tolerance

TR coexpressing both genes tolerated Cd and TCE very well. TR with single genes were weakly tolerant

Zhang and Liu (2011)

The combined expression of both genes showed cross tolerance to the complex of heavy metals and organic pollutants (Continued)

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TABLE 6.1 (Continued) Product and Heavy Metal Phytochelatin Cd, As

Gene Transferred and Source AsPCS1 and YCF1 Allium sativum and yeast Sachcharomyces cerevisiae

Transformed Plant A. thaliana

In Vitro Test

Results and Comments

Reference

Two day seedlings grown on 1/2 MS and germination medium with Cd and As for 14 days

TR with both genes had longest roots and higher accumulation of Cd (2- to 10-fold) and As (2- to 3-fold) than single gene TR or WT

Guo et al. (2012)

Simultaneous expression of both genes increased tolerance to and accumulation of Cd and As. Metal transporter PvACR3 As Pteris vittata

A. thaliana

Three-day-old seedlings of Arabidopsis were grown on 1 /2 MS agar with As(III) and As(V) for 7 or 15 days

Enhanced tolerance in TR. Chen et al. (2013) As was reduced in roots and increased in shoots. TR in soil with 10 ppm As (V) accumulated 7.5-fold more As than WT TR seeds germinate and grow in 80 μM As(III) or 1200 μM As(V), lethal to WT seeds. Potentials to transfer the As transporter gene fast growing high biomass species

Metallothionein Cu

Glutathione Cd, Zn, Cu

ThMT3 Salix matsudana Tamarix hispida Agrobacterium mediated transformation of seeds

Glutamylcysteine synthetase Streptococcus thermophilus

Beta vulgaris

In vitro shoots of 3 cm height transferred to 1/2 MS with 0 to 50 μM CuSO4 for 4 weeks

Yang et al. ThTM3 increased Cu tolerance and nitric oxide (2015) production which induced adventitious roots 50 μM Cu completely inhibited rooting in WT; induced adventitious roots in TR

One week TR seedlings grown on MS medium with 0 200 μM of Cd21, Zn21, or Cu21 alone and in combinations

TR accumulated more Cu, Liu et al. Zn, Cd in shoots than WT. (2015) TR tolerated combinations of 50 μM of Cd-Zn, Cd-Cu, Zn-Cu and Cd-Zn-Cu. Tolerance is likely due to overproduction of glutathione and phytochelatins (Continued)

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TABLE 6.1 (Continued) Product and Heavy Metal Metal tolerance/ transporter Cd and As

Gene Transferred and Source

Transformed Plant

OsMTP1 Oryza sativa

Nicotiana tabacum

In Vitro Test

Results and Comments

Reference

Seeds on MS agar. 7 d seedlings to MS medium with 100 μM CdCl2 & 200 μM Na2HAsO4

Reduced Cd induced phytotoxic stress; increased levels of vacuolar thiols. Higher biomass 2.2- to 2.8-fold & Cd content 1.96- to 2.22fold

Das et al. (2016)

Moderate tolerance and accumulation of As Transporter Hg

PaMerT Pseudomonas alcaligenes

A. thaliana Agrobacterium mediated transformation by floral dip

Seeds cultured on MS agar medium with 0 to 40 μM L21 HgCl2.

Enhanced tolerance to HgCl2 and transcript levels of antioxidant enzymes in TR

Xu et al. (2017)

Prevented oxidative damage to the plant; gene is likely associated with vacuole Transporter

SaNramp6

Cd

Sedum alfredii

A. thaliana

30 d seedlings of A. thaliana were treated with 50 μM CdCl2

In tobacco, Nicotiana tabacum, Pavlikova et al. (2004) tested four genetically modified lines with the CUP1 transgene encoding a yeast metallothionein gene, for the accumulation of Cd, Zn, and Ni on a sand nutrient medium. Of the four gene constructs used, the Cd levels in the shoots was increased by 90% compared with the nontransformed lines by the HisCUP line. The presence of the CUP1 metallothionein gene alone did not increase the accumulation of Cd significantly over the controls. However, Hasegawa et al. (1997) overexpressed CUP1 in cauliflower to give a 16-fold higher Cd accumulation. Kra¨mer et al. (1996) earlier showed that free histidine is a metal chelator for Ni. The CUP1

TR overexpressing the gene had higher Cd concentrations in the shoots. The SaNramp6 gene is localized in the plasma membrane

Chen et al. (2017)

metallothionein gene with a polyhistidine tail showed a significant increase in Cd and the GUS construct with polyhistidine for Ni. These results on sand nutrient medium are an indication of the phytoremediation potential from contaminated soil. While Zn was not translocated to the shoots, Ni content was significantly high in the shoots with the HisGUS line. Another metallothionein gene, PiMT1, was cloned from the fungus Paxillus involutus by Bellion et al. (2007) and the ectomycorrhizal fungus Hebeloma cylindrosporum was transformed by agrobacterium mediated transformation. The constitutive overexpression of the PiMT1 gene resulted in a higher tolerance to Cu in the fungus.

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TABLE 6.2 Hydroponic and Soil Experiments to Assess Transgenic Plants After Transformation to Enhance Their Phytoremediation Capacity in Selected Experiments (TR Transgenic, WT Wild Type) Product and Gene Transferred and Heavy Metal Source

Transformed Plant

ATP sulfurylase

Adenosine triphosphate sulfurylase (APS) from A. thaliana

γ-Glutamylcysteine synthetase

γ-Glutamyl-cysteine synthetase (ECS) from E. coli

Glutathione synthetase

Glutathione synthetase (GS) from E. coli and glutathione synthetase (GS)

Metal transporter Pb and Cd

YCF1 S. cerevisiae

Phytochelatin TaPCS1 Pb Triticum aestivum

Hydroponics, Soil Experiments

Results and Comments

Reference

B. juncea

Soil from 0 25 cm was collected from the contaminated site and mixed with normal top soil. Plot size 3.3 m 3 1 m for 3 TR lines, 1 WT, and 1 base plot

APS transgenic line accumulated 4.3-fold, ECS transgenic line accumulated 2.8-fold, GS transgenic line accumulated 2.3-fold. GS lines tolerated the contaminated soil better. TR lines accumulated more Se in shoots than WY

Ban˜uelos et al. (2005)

A. thaliana

TR seedlings germinated on 1/2 MS were transferred to fine gravel soaked in 1/2 MS medium with 0.75 mM Pb(II) or 70 μM Cd (II) for 3 weeks before harvesting

Plants overexpressing YCF1 have enhanced tolerance to Pb(II) and Cd (II)

Song et al. (2010)

Populus tremula x P. tremuloides cv. Etrepole

YCF1 is an efficient mechanism to transport Pb and Cd into the vacuoles and enable detoxification

Hydroponic: in vitro rooted plants in plastic vessels with sterile substrate; 0 to 3 mM Pb (NO3)2 1 Hoagland solution

No difference in Pb levels in TR and Couselo et al. WT; total biomass and Pb (2010) accumulation higher in TR

Plants also grown in Pb/Zn contaminated mine soil

Similar results with mine soil 3 mM of Pb was toxic to TR & WT Pb accumulation higher in roots 92 94%. Phytoremediation potential is determined by physicochemical properties of soil

Glutathione transferases Hg and TCE

pKHCG GST and P450 2E1

Medicago sativa

Seeds of TR and WT were grown on MS medium with HgCl2/TCE for 3 weeks and fresh weights, Hg, and TCE determined. Pot experiments with 1 kg soil for 3 weeks also conducted

Enhanced resistance to the toxic effects of Hg/TCE by the TR. Accumulation was 3- to 4.2-fold higher for Hg and 2.1- to 4.0-fold higher for TCE than WT plants Coexpression of both genes had no effect on growth of TR.

Zhang et al. (2013)

Metal transporter Cd

ScYCF1 Yeast Sachcharomyces cerevisiae

Phytochelatin PtPCS Cd Populus tormentosa

Populus alba x P. tremula var. grandulosa sterile

TR seedlings grown in hydroponics with 1 ppm Cd for 4 weeks. Plantlets grown in mine tailing soil in pots (Cd tolerance test) for 2 weeks and for 60 days with Cd for root survival Field test: in pots with diluted mine tailing soil (6:1) for 193 days

TR had increased growth, reduced toxicity symptoms, and high Cd levels in the shoots. Tolerant to multiple toxic metal(loids) Root system of TR was extensive; also accumulated Cd, Zn, and Pb in the roots. Bioconcentration in the field was lower than in hydroponics

Shim et al. (2013)

Tobacco Agrobacterium mediated leaf disk transfer

TR and WT transplanted to pots with perlite and vermiculite (1:1) treated with 90 μM CdCl2 for 30 days

Better growth and tolerance by TR to Cd. 1.7- to 3.0-fold higher Cd in roots; 1.24- to 1.28-fold higher in leaves

Chen et al. (2015)

Transfer coefficient lower in TR than WT. PtPCS is involved in Cd tolerance and not transport Glutathione Cd

Glutamyl-cysteine synthetase bacterial

Poplar: P. tremula x P. alba

Transgenics were tolerant to 100 μM He et al. In vitro propagated plants after (2015) rooting were treated in Hoagland Cd21 than WT solution with 0 and 100 μM Cd21 TR had higher Cd uptake, high in a growth chamber transcript levels of genes involved in Cd transport and detoxification, greater accumulation in the shoots than WT

Transporter Methyl Hg

MerC bacterial

Arabidopsis thaliana

Seedlings grown in 1/10 Hoagland solution in 1% (w/v) agar for 12 days in a growth chamber

MerC was able to transport methyl mercury and also Hg(II) and Cd(II) Translocation efficiency of Hg from roots to shoots needs to be improved

Sone et al. (2017)

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Pomponi et al. (2006) overexpressed the phytochelatin synthase gene (PCS1) from A. thaliana in the nonaccumulator N. tabacum, which was transformed with the rolB oncogene from Agrobacterium rhizogenes. Tobacco plants with the rolB genes easily form root cultures, which is an ideal system to study heavy metal tolerance. The overexpression of the phytochelatin synthase gene increased Cd21 tolerance in the roots of the tobacco plants. This was further enhanced when the precursor of the phytochelatin synthase gene, reduced glutathione, was added to the culture medium. Thus, the increase of Cd21 tolerance and accumulation by plants overexpressing the phytochelatin synthase gene was directly related to the availability of the precursor glutathione. However, the root to shoot translocation of Cd21 was not enhanced by the phytochelatin synthase gene. The PCS1 gene from wheat, Triticum aestivum, transformed into Nicotiana glauca also enhanced tolerance to Cd21 and Pb21 and accumulation of Pb21 (Gilbert et al., 2003). However, hypersensitivity to Cd21 was reported in Arabidopsis overexpressing AtPCS1 (Lee et al., 2003; Li et al., 2004). These differences in response, according to Pomponi et al. (2006), could be attributed to much higher concentrations of the phytochelatins in Arabidopsis compared with tobacco and may also be linked to differences in physiology between the two species, suggesting a species-specific response. The studies by Couselo et al. (2010) on the expression of the phytochelatin synthase gene in Populus did not improve their response to stress. This is in contrast to results obtained with Nicotiana glauca, which accumulated sixfold higher Pb than the wild type grown in similar soil (Martı´nez et al., 2006). Thus, the negative outcome with Populus by Couselo et al. (2010) emphasizes the need to follow up in vitro studies at the field level and also to look at other strategies to enhance transgenic approaches to phytoremediation.

Chen et al. (2015) cloned the gene encoding for phytochelatin synthase from poplar (Populus tomentosa), a woody plant known to accumulate high levels of heavy metals, particularly Cd. This gene was transferred into tobacco using Agrobacterium mediated leaf disk transformation. After treatment of the wild type and transgenic plants, the latter accumulated 1.7- to 3.0-fold higher Cd levels in the roots and 1.24 to 2.28 higher Cd levels in the shoots. The transfer of Cd from roots to shoots, however, was higher in the wild type plants. The authors concluded that the phytochelatin synthase gene encodes a functional phytochelatin synthase apparently involved in Cd tolerance and accumulation but not in the transport of Cd. In transgenic plants transformed with the PCS gene, hypersensitivity is often observed to heavy metals. This could be due to expression of phytochelatins at supraoptimal levels relative to glutathione (GSH) levels, which limits the synthesis of phytochelatins (Guo et al., 2012, also see Pomponi et al., 2006). Transporting the heavy metals into the plant cell is not sufficient by itself, they need to be isolated in the vacuoles to prevent their interference with cellular processes, which is reflected as tolerance to the pollutants (see Tong et al., 2004). In this context Guo et al. (2012) transformed A. thaliana with AtPCS1 and YCF1, genes for metal chelation by thiols and transfer to vacuoles, respectively, for detoxification and tolerance to Cd and As. The dual-gene transgenic lines accumulated 2- to 10-fold Cd/arsenite and 2- to 3-fold arsenate over WT or plants expressing only one of these genes. Detoxification and tolerance to heavy metals require both the transfer of the heavy metals into the plant cell and subsequent isolation by transport across the vacuolar cell membrane into the vacuole (Song et al., 2010). The YCF1 gene isolated from Saccharomyces cerevisiae is one of the well-characterized transporter genes (Li et al., 1997; Tong et al., 2004). Song et al. (2003) found that YCF1 enhanced tolerance and accumulation of Cd and Pb in

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6.3 THE ROLE OF IN VITRO STUDIES

the shoots of A. thaliana, even at very low expression levels. Another gene in the ABC transporter family is AtATM3 found in the mitochondrial membrane of A. thaliana. Bhuiyan et al. (2011) transformed B. juncea with the YCF1 gene to improve heavy metal tolerance and accumulation. The transgenic plants had 1.5- to 2.5-fold higher Cd and Pb levels than the control wild type seedlings. The authors attributed this to the increased expression of glutathione synthetase and phytochelatin synthase brought about by the overexpression of AtATM3. In a study with sterile Populus plants, transformed with the YCF1 gene, Shim et al. (2013) conducted hydroponics and pot experiments with soil from an abandoned mining site with Cd and other heavy metals. The transgenic poplar plants in the hydroponic medium with 1 mg/kg Cd accumulated 23 mg/kg Cd in 4 weeks while the wild type accumulated only 9 mg/kg. However, field experiments had a bioconcentration factor of 0.3 for Cd, 0.9 for Zn, 0.2 for As, and 1.9 for Pb. The authors attribute this to the presence of toxic metal(loid)s in the field soil compared with the single metal in the hydroponic solution. The plants develop an extensive root system and hence are suitable for phytostabilization of contaminated sites, by preventing soil erosion, dispersion of the pollutants, and phytoextraction by virtue of their life span of 30 years (Shim et al., 2005). In view of the low field accumulation of Cd by the single YCF1 gene, the authors suggest that gene stacking for uptake, translocation, and chelation can enhance the phytoremediation capacity at the field level. Genes are available for different physiological processes and if these can be coexpressed to function synergistically, the performance of the YCF1 gene can be enhanced to perform on contaminated sites (Shim et al., 2013). Liu et al. (2015) used a novel enzyme from Streptococcus thermophiles StGCS-GS, involved in glutathione biosynthesis, to transform sugar

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beet (Beta vulgaris), a high biomass and energy crop. In vitro studies were conducted to assess the heavy metal tolerance of the transgenic plants by treating three transgenic lines and the wild type in MS medium (Murashige and Skoog, 1962) supplemented with 0 200 μM of Cd21, Zn21, and Cu21, separately and in combination at 50 μM of each of the three metals, for 3 weeks. Heavy metal tolerance was estimated from the fresh weight and root length. In addition, a hydroponics experiment was also conducted with the same concentrations of heavy metals. The accumulation of heavy metal and the glutathione and phytochelatins contents in the tissues were also determined. One transgenic line, which expressed the highest StGCSGS, accumulated an increased ion content of Cd, Zn and Cu in the shoots. The glutathione levels were significantly higher in the StGCSGS transgenic lines than in the wild type, while the phytochelatins levels were nearly the same. The treatment with combinations of the three ions caused severe damage to all the tested transgenic lines. However, the authors report that the overexpression of the StGCS-GS improved the tolerance and increased the accumulation of the complex of metals. The authors conclude that overexpression of the StGCS-GS gene can increase the accumulation of Cd, Zn, and Cu in sugar beet and tolerance to heavy metals without affecting their growth. Phytochelatins, whose precursor is glutathione, bind to heavy metals in the cytosol to form a nontoxic protein complex, which can be translocated into vacuoles for isolation. An important enzyme in glutathione biosynthesis is γ-glutamyl-cysteine synthetase (γ-ECS) (Arisi et al., 2000). Transgenic poplar lines with the γ-ECS enzyme in the cytosol had a 30-fold increase in this enzyme’s activity compared to untransformed controls and up to 3.5-fold increase in glutathione in the leaves (Arisi et al., 2000). He et al. (2015) used two transgenic lines from transformed P. tremula x P. alba overexpressing γ-ECS enzyme to produce micropropagated

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plantlets that were rooted and transferred to aerated modified Hoagland solution for hydroponic culture for 80 days. Two groups of plants were tested with 0 and 100 μM Cd21. The net Cd21 influx, tolerance to Cd, and the regulation of the genes involved in Cd21 transport were determined in the transgenic and wild type poplars. The transgenics had higher Cd21 uptake rates and elevated levels of the genes involved in Cd21 transport and detoxification than the wild type. The accumulated Cd21 ions were in the shoots of the transgenics and GSH and oxidized GSH were in the roots and leaves. The authors conclude that the transgenics could tolerate up to 100 μM Cd21 probably due to the GSH mediated induction of the transcription of genes involved in Cd transport and detoxification. Transformation with single genes that show positive results in in vitro experiments are not always manifested as field performances. Thus, more elucidation of the biochemical and physiological response at the plant level is necessary to realize the potential of transgenics in phytoremediation.

6.4 FUTURE PERSPECTIVES 6.4.1 Risks Associated With Transgenic Plants Over the years the release of transgenic organisms in the environment has been a cause of concern, particularly in Europe. Transgenic plants, in general, face opposition to their release into the environment from public and environmental lobbies due to concerns of safety to human health and impacts on the environment. These concerns should be carefully addressed to facilitate the use of transgenic plants for phytoremediation. A major concern is the gene flow from transgenics to genetically related wild type or crop relatives (Fig. 6.2). Gene flow from plants

engineered to tolerate and uptake heavy metals to related species would give them a selective edge to spread on similar and possibly to nonpolluted sites (Glass, 2005; Davison, 2005). This could potentially confer a selective advantage over native species to tolerate heavy metals and outcompete other species in the environment, leading to invasive weedy species and eventually a threat to biodiversity. Species that are recruited for transformation are either plants already in use (e.g., Brassica juncea) or wild species without commercial or agricultural importance. Since most domesticated plants are derived from their wild relatives, the chances of a transgenic plant of commercial utility successfully interbreeding with their wild weedy relatives is high (Glass, 2005). Thus, the immediate concern is whether the enhancement of hyperaccumulation by genetic modification of a species would further bestow on it an advantage to survive and outcompete other species in the wild. Another major concern for field planting of transgenic plants is their ability to interbreed with related crop species through cross pollination and transfer their metal uptake abilities to crops, which can then enter the human food chain. Mustard is a popular oil crop in India where the seed is used as a spice and the oil extracted from the seeds is used as cooking oil. Thus, if the hyperaccumulated metal is sequestered in the seed and such a transgenic species outcrosses with the cultivated crop it would be impossible to reverse the process. The extent of concern is also different between countries. The regulatory framework of the United States is based on the evaluation of the product while the European Commission is concerned with evaluation of the process and the application of the precautionary principle (Nicolia et al., 2014). Based on these two systems other countries have developed their own regulatory frameworks (Ramessar et al., 2008). This also explains the lack of consensus among countries on the release of transgenic plants

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6.4 FUTURE PERSPECTIVES

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FIGURE 6.2 Gene flow from transgenic plants.

into the environment. The absence of uniformity in the regulations from country to country and the shortcomings in the dialogue between scientists and laymen has contributed to the health and environmental concerns of transgenics in the public mind (Nicolia et al., 2014). The EU funded over 50 research programs from 2001 to 2010 with a budget of 200 million euros, to address public concerns on the safety of transgenic crops and the overall conclusion of these studies is that the use of biotechnology and genetically engineered plants, per se, does not show higher risks than classical breeding methods (European Commission, 2010). A similar conclusion was reached by Nicolia et al. (2014) by reviewing the scientific literature for 10 years: research has not shown any significant hazard directly connected with the use of genetically modified crops.

Crop plants in agriculture were initially engineered with genes for resistance against insects, virus and herbicides, which met with heavy resistance from environmental groups and consumers, particularly in Europe. Many of these potential concerns would not apply to transgenics in phytoremediation, since these would not enter the human food chain.

6.4.2 Overcoming the Risks From Transgenes Several approaches are available to address the risks associated with the release of transgenic plants for heavy metal uptake from the soil, particularly the contamination of the native gene pool and heavy metals entering the food chain (Fig. 6.3).

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FIGURE 6.3 Containment of transgenic phytoremediator species.

Transgenics can be created with plant species that are sterile or propagate vegetatively. The sterile species can be propagated by in vitro micropropagation methods for largescale planting to remediate contaminated sites. Alternatively, an additional transformation of the transgenic species to render it sterile and subsequent in vitro propagation can also provide a viable solution. Using self-pollinated transgenic species for phytoremediation can also avoid unintended gene flow (Wu et al., 2010). However, care should be taken for careful assessment of the degree of self-pollination since many self-pollinated species have a very low level of outcrossing. The unintentional transfer of genes can also be prevented by using species with a long generation interval (such as perennial trees) to create transgenics, which can be harvested before the onset of flowering. Wu et al. (2010) have suggested the creation of infertile polyploid plants using allopolyploids and triploids for hybridization. Polyploids can also be created by somatic hybridization or cell fusion. Polyploids are usually bigger and hence have a stronger transpiration flow from roots to shoots to facilitate the transport of metals (Wu et al., 2010). The contamination of large areas of farmland by heavy metals needs remediation. Unlike

mining sites, these cannot be planted with conventional hyperaccumulators since this would pose problems for food security. Wu et al. (2010) have suggested that transgenic crop plants capable of hyperaccumulating heavy metals can be used provided that the heavy metals do not accumulate in the edible parts of the plant. Such crop hyperaccumulators are wheat, brassica, maize, and rice. These species need stringent screening of their plant parts to ensure that metals do not accumulate in the grains or seeds. Contamination of human and animal food can be avoided by using species that are not consumed by humans or animals, e.g., species that produce secondary metabolites that are repellent to animals and also insects. Grazing of the transgenic plants by animals is a risk that can be prevented by labeling and fencing the polluted sites where phytoremediation is in progress. Several gene containment approaches are discussed by Davison (2005) to mitigate the risk of gene escape into the environment from transgenic plants. These are experimental approaches tested on model plants under controlled conditions. Davison (2005) has suggested designing plants for phytoremediation for conditional suicide. For example, plants designed for mercury remediation would depend for their survival on

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REFERENCES

the presence of mercury; once it is depleted or if the plant escapes to a mercury free environment, this would activate suicide genes in the plant. Engineering genetic incompatibility between otherwise sexually reproducing populations can contain the escape of genes to the wild (Maselko et al., 2017). This was demonstrated in Saccharomyces cerevisiae where the lethal overexpression of actin results when the transgenic variety strain is crossed with the wild type.

6.4.3 The Way Forward Introducing transgenic plants for phytoremediation needs to overcome the public concerns and the regulatory framework in individual countries. The major concerns are the irreversibility of introducing a foreign gene into a new species; gene flow into related species, particularly crops, by which they can enter the food chain; and the threat to biodiversity. These concerns can be effectively addressed by transforming species that are sterile and propagated vegetatively. This would effectively prevent gene flow of heavy metal uptake capabilities into related species. The advantages of phytoremediation over conventional methods also needs emphasis. Conventional methods use physical excavation and washing of soils and chemical methods to precipitate the heavy metals in contrast to phytoremediation, which is environmentally benign and inflicts the least disturbance to the environment. Transformation with a single gene has not always been effective. It is necessary to look at the process of absorption, translocation from roots to shoots, and sequestration in the cell vacuoles and consider gene stacking. The same transgene expressed in different target species have produced species-specific response such as in Arabidopsis and Nicotiana, due to differences in plant physiology (Lee et al., 2003; Li

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et al., 2004; Pomponi et al., 2006). Transgenics thus need a detailed evaluation of how the transgene is expressing itself in a new genomic environment. When transgenes are sourced from plants, performing transformations within the same family can reduce the problems of expression. The mechanisms of metal translocation from the roots to the shoots can also differ between species. The evaluation of these preliminary steps of transforming a plant is possible only through in vitro methods, which provide a homogenous environment and replications of the experiment. This should be followed by hydroponic experiments and contained field evaluation before release of the transgenic phytoremediators for field applications.

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FURTHER READING

Sarma, H., 2011. Metal hyperaccumulation in plants: a review focusing on phytoremediation technology. J. Environ. Sci. Technol. 4 (2), 118 138. Silver, S., Phung, L.T., 2005. A bacterial view of the periodic table: genes and proteins for toxic inorganic ions. J. Industrial Microbiol. Biotech. 32 (11 12), 587 605. Shim, D., Kim, S., Choi, Y.I., Song, W.Y., Park, J., Youk, E. S., et al., 2005. A bacterial view of the periodic table: genes and proteins for toxic inorganic ions. J. Ind. Microbiol. Biotechnol. 32 (11-12), 587 605. Shim, D., Kim, S, Choi, Y.-I., Song, W.-Y., Park, J., Youk, E. S., et al., 2013. Transgenic poplar trees expressing yeast cadmium factor 1 exhibit the characteristics necessary for the phytoremediation of mine tailing soil. Chemosphere 90 (4), 1478 1486. Sone, Y., Uraguchi, S., Takanezawa, Y., Nakamura, R., PanHou, H., Kiyono, M., 2017. A movel role of MerC in methylmercury transport and phytoremediation of methylmercury contamination. Biol. Pharmaceut. Bull. 40 (7), 1125 1128. Song, W.Y., Sohn, E.J., Martinoia, E., Lee, Y.J., Yang, Y.Y., Jasinski, M., et al., 2003. Engineering tolerance and accumulation of lead and cadmium in transgenic plants. Nature Biotech 21 (8), 914 919. Song, W.Y., Park, J., Mendoza-Co´zatl, D.G., SuterGrotemeyer, M., Shim, D., Ho¨rtensteiner, S., et al., 2010. Arsenic tolerance in Arabidopsis is mediated by two ABCC-type phytochelatin transporters. Proc. Natl Acad. Sci. 107 (49), 21187 21192. Tavakkoli, E., Fatehi, F., Rengasamy, P., McDonald, G.K., 2012. A comparison of hydroponic and soil-based screening methods to identify salt tolerance in the field in barley. J. Exp. Botany 63 (10), 3853 3867. Tong, Y.P., Kneer, R., Zhu, Y.G., 2004. Vacuolar compartmentalization: a second-generation approach to engineering plants for phytoremediation. Trends Plant Sci. 9 (1), 7 9. Utmazian, M.N.D.S., Wieshammer, G., Vega, R., Wenzel, W. W., 2007. Hydroponic screening for metal resistance and accumulation of cadmium and zinc in twenty clones of willows and poplars. Environ. Pollut. 148 (1), 155 165. Van Aken, B., 2008. Transgenic plants for phytoremediation: helping nature to clean up environmental pollution. Trends Biotechnol. 26 (5), 225 227. Verret, F., Gravot, A., Auroy, P., Leonhardt, N., David, P., Nussaume, L., et al., 2004. Overexpression of AtHMA4 enhances root-to-shoot translocation of zinc and cadmium and plant metal tolerance. FEBS Lett. 576 (3), 306 312. Watson, C., Pulford, I.D., Riddell-Black, D., 2003. Development of a hydroponic screening technique to assess heavy metal resistance in willow (Salix). Int. J. Phytoremediation 5 (4), 333 349. Wu, G., Kang, H., Zhang, X., Shao, H., Chu, L., Ruan, C., 2010. A critical review on the bio-removal of hazardous

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heavy metals from contaminated soils: issues, progress, eco-environmental concerns and opportunities. J. Hazard. Mater. 174 (1-3), 1 8. Xu, J., Chai, T., Zhang, Y., Lang, M., Han, L., 2009. The cation-efflux transporter BjCET2 mediates zinc and cadmium accumulation in Brassica juncea L. leaves. Plant Cell Rep. 28 (8), 1235 1242. Xu, S., Sun, B., Wang, R., He, J., Xia, B., Xue, Y., et al., 2017. Overexpression of a bacterial mercury transporter MerT in Arabidopsis enhances mercury tolerance. Biochem. Biophys. Res. Commun. 490 (2), 528 534. Yadav, S.K., 2010. Heavy metals toxicity in plants: an overview on the role of glutathione and phytochelatins in heavy metal stress tolerance of plants. S. Afr. J. Bot. 76 (2), 167 179. Yang, J., Li, K., Zheng, W., Zhang, H., Cao, X., Lan, Y., et al., 2015. Characterization of early transcriptional responses to cadmium in the root and leaf of Cd-resistant Salix matsudana Koidz. BMC Genomics 16 (1), 705. Zhang, Y., Liu, J., 2011. Transgenic alfalfa plants coexpressing glutathione S-transferase (GST) and human CYP2E1 show enhanced resistance to mixed contaminates of heavy metals and organic pollutants. J. Hazard. Mater. 189 (1-2), 357 362. Zhang, Y., Liu, J., Zhou, Y., Gong, T., Wang, J., Ge, Y., 2013. Enhanced phytoremediation of mixed heavy metal (mercury) organic pollutants (trichloroethylene) with transgenic alfalfa co-expressing glutathione S-transferase and human P450 2E1. J. Hazard. Mater. 260, 1100 1107.

Further Reading Dhankher, O.P., Li, Y., Rosen, B.P., Shi, J., Salt, D., Senecoff, J.F., et al., 2002. Engineering tolerance and hyperaccumulation of arsenic in plants by combining arsenate reductase and γ-glutamylcysteine synthetase expression. Nat. Biotechnol. 20 (11), 1140. Fingerman, M. (Ed.), 2016. Bioremediation of Aquatic and Terrestrial Ecosystems. CRC Press, Taylor and Francis, Boca Raton. Hejabi, A.T., Basavarajappa, H.T., Karbassi, A.R., Monavari, S.M., 2011. Heavy metal pollution in water and sediments in the Kabini River, Karnataka, India. Environ. Monitor. Assess. 182 (1-4), 1 13. Henry, R.B., Liu, J., Choudhuri, S., Klaassen, C.D., 1994. Species variation in hepatic metallothionein. Toxicol. Lett. 74 (1), 23 33. Krystofova, O., Zitka, O., Krizkova, S., Hynek, D., Shestivska, V., Adam, V., et al., 2012. Accumulation of cadmium by transgenic tobacco plants (Nicotiana tabacum L.) carrying yeast metallothionein gene revealed by electrochemistry. Int. J. Electrochem. Sci 7, 886 907.

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Lee, Y., 2003. Engineering tolerance and accumulation of lead and cadmium in transgenic plants. Nat. Biotechnol. 21 (8), 914. Lee, Y., 2013. Transgenic poplar trees expressing yeast cadmium factor 1 exhibit the characteristics necessary for the phytoremediation of mine tailing soil. Chemosphere 90 (4), 1478 1486. McIntyre, T., 2003. Phytoremediation of heavy metals from soils. Phytoremediation. Springer, Berlin Heidelberg, Germany, pp. 97 123. Meagher, R.B., 2000. Phytoremediation of toxic elemental and organic pollutants. Curr. Opin. Plant Biol. 3 (2), 153 162. Meister, A., Anderson, M.E., 1983. Glutathione. Annu. Rev. Biochem. 52 (1), 711 760. Memon, A.R., Schro¨der, P., 2009. Implications of metal accumulation mechanisms to phytoremediation. Environ. Sci. Pollut. Res. 16 (2), 162 175.

Palmiter, R.D., 1994. Regulation of metallothionein genes by heavy metals appears to be mediated by a zincsensitive inhibitor that interacts with a constitutively active transcription factor, MTF-1. Proc. Natl Acad. Sci. 91 (4), 1219 1223. Schro¨der, P., Collins, C., 2002. Conjugating enzymes involved in xenobiotic metabolism of organic xenobiotics in plants. Int. J. Phytoremediation 4 (4), 247 265. Song, W.Y., Sohn, E.J., Martinoia, E., Lee, Y.J., Yang, Y.Y., Jasinski, M., et al., 2012. A comparison of hydroponic and soil-based screening methods to identify salt tolerance in the field in barley. J. Exp. Bot. 63 (10), 3853 3867. ´ ´ ´ Wawrzynski, A., Kopera, E., Wawrzynska, A., Kaminska, J., Bal, W., Sirko, A., 2006. Effects of simultaneous expression of heterologous genes involved in phytochelatin biosynthesis on thiol content and cadmium accumulation in tobacco plants. J. Exp. Bot. 57 (10), 2173 2182.

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