Application of hairy roots for phytoremediation: what makes them an interesting tool for this purpose?

In recent years, hairy roots (HRs) have been successfully used as research tools for screening the potentialities of different plant species to tolerate, accumulate, and/or remove environmental pollutants, such as PCBs, TNT, pharmaceuticals, textile dyes, phenolics, heavy metals, and radionuclides. This is in part due to several advantages of this plant model system and the fact that roots have evolved specific mechanisms to deal with pollutants because they are the first organs to have contact with them. In addition, by using HRs some metabolic pathways and enzymatic catalyzed reactions involved in pollutants detoxification can be elucidated as well as the mechanisms of uptake, transformation, conjugation, and compartmentation of pollutants in vacuoles and/or cell walls, which are important detoxification sites in plants. Plant roots also stimulate the degradation of contaminants by the release of root exudates and oxido-reductive enzymes, such as peroxidases (Px) and laccases, that are associated with the removal of some organic pollutants. HRs are also considered good alternatives as enzyme sources for remediation purposes. Furthermore, application of genetic engineering methods and development of microbe-assisted phytoremediation are feasible strategies to enhance plant capabilities to tolerate, accumulate, and/or metabolize pollutants and, hence, to create or find an appropriate plant system for environmental cleanup. The present review highlights current knowledge, recent progress, areas which need to be explored, and future perspectives related to the application and improvement of the efficiency of HRs for phytoremediation research.     

Hairy root research: recent scenario and exciting prospects

High stability of the production of secondary metabolites is an interesting characteristic of hairy root cultures. For 25 years, hairy roots have been investigated as a biological system for the production of valuable compounds from medicinal plants. A better understanding of the molecular mechanism of hairy root development, which is based on the transfer of Agrobacterium rhizogenes T-DNA into the plant genome, has facilitated its increasing use in metabolic engineering. Hairy roots can also produce recombinant proteins from transgenic roots, and thereby hold immense potential for the pharmaceutical industry. In addition, hairy roots offer promise for phytoremediation because of their abundant neoplastic root proliferation. Recent progress in the scaling-up of hairy root cultures is making this system an attractive tool for industrial processes.     

Recent Findings on the Phytoremediation of Soils Contaminated with Environmentally Toxic Heavy Metals and Metalloids Such as Zinc,Cadmium, Lead,and Arsenic

Due to their immutable nature, metals are a group of pollutants of much concern. As a result of human activities such as mining and smelting of metalliferous ores, electroplating, gas exhaust, energy and fuel production, fertilizer and pesticide application, etc., metal pollution has become one of the most serious environmental problems today. Phytoremediation, an emerging cost-effective, non-intrusive, and aesthetically pleasing technology, that uses the remarkable ability of plants to concentrate elements and compounds from the environment and to metabolize various molecules in their tissues, appears very promising for the removal of pollutants from the environment. Within this field of phytoremediation, the utilization of plants to transport and concentrate metals from the soil into the harvestable parts of roots and above-ground shoots, i.e., phytoextraction, may be, at present, approaching commercialization. Improvement of the capacity of plants to tolerate and accumulate metals by genetic engineering should open up new possibilities for phytoremediation. The lack of understanding pertaining to metal uptake and translocation mechanisms, enhancement amendments, and external effects of phytoremediation is hindering its full scale application. Due to its great potential as a viable alternative to traditional contaminated land remediation methods, phytoremediation is currently an exciting area of active research.     

Phragmites australis — a helophytic grass — can establish successful partnership with phenol-degrading bacteria in a floating treatment wetland

Helophytic plants contribute significantly in phytoremediation of a variety of pollutants due to their physiological or biochemical mechanisms. Phenol, which is reported to have negative/deleterious effects on plant metabolism at concentrations higher than 500 mg/L, remains hard to be removed from the environmental compartments using conventional phytoremediation procedures. The present study aims to investigate the feasibility of using P. australis (a helophytic grass) in combination with three bacterial strains namely Acinetobacter lwofii ACRH76, Bacillus cereus LORH97, and Pseudomonas sp. LCRH90, in a floating treatment wetland (FTW) for the removal of phenol from contaminated water. The strains were screened based on their phenol degrading and plant growth promoting activities. We found that inoculated bacteria were able to colonize in the roots and shoots of P. australis, suggesting their potential role in the successful removal of phenol from the contaminated water. Pseudomonas sp. LCRH90 dominated the bacterial community structure followed by A. lowfii ACRH76 and B. cereus LORH97. The removal rate was significantly high when compared with the individual partners, i.e., plants and bacteria separately. The plant biomass, which was drastically reduced in the presence of phenol, recovered significantly with the inoculation of bacterial consortia. Likewise, highest reduction in chemical oxygen demand (COD), biochemical oxygen demand (BOD), and total organic carbon (TOC) is achieved when both plants and bacteria were employed. The study, therefore, suggests that P. australis in combination with efficient bacteria can be a suitable choice to FTWs for phenol-degradation in water.     

A Hydroponic System for Growing Gnotobiotic Vs. Sterile Plants to Study Phytoremediation Processes

In some phytoremediation studies it is desirable to separate and define the specific contribution of plants and root-colonizing bacteria towards contaminant removal. Separating the influence of plants and associated bacteria is a difficult task for soil root environments. Growing plants hydroponically provides more control over the biological factors in contaminant removal. In this study, a hydroponic system was designed to evaluate the role of sterile plant roots, rhizodeposition, and root-associated bacteria in the removal of a model contaminant, phenol. A strain of Pseudomonas pseudoalcaligenes that grows on phenol was inoculated onto plant roots. The introduced biofilm persisted in the root zone and promoted phenol removal over non-augmented controls. These findings indicate that this hydroponic system can be a valuable tool for phytoremediation studies that investigate the effects of biotic and abiotic factors on pollution remediation.     

Bioreactor technology: a novel industrial tool for high-tech production of bioactive molecules and biopharmaceuticals from plant roots

Plants are the richest source for different bioactive molecules. Because of the vast number of side effects associated with synthetic pharmaceuticals, medical biotechnologists turned to nature to provide new promising therapeutic molecules from plant biofactories. The large-scale availability of the disease- and pesticide-free raw material is, however, restricted in vivo. Many bioactive plant secondary metabolites are accumulated in roots. Engineered plants can also produce human therapeutic proteins. Vaccines and diagnostic monoclonal antibodies can be won from their roots, so that engineered plants hold immense potential for the biopharmaceutical industry. To obtain sufficient amounts of the plant bioactive molecules for application in human therapy, adventitious and hairy roots have to be cultured in in vitro systems. High-tech pilot-scale bioreactor technology for the establishment of a long-term adventitious root culture from biopharmaceutical plants has recently been established. In this review, I briefly discuss a technology for cultivating bioactive molecule-rich adventitious and hairy roots from plants using a high-tech bioreactor system, as well as the principles and application of genome-restructuring mechanisms for plant-based biopharmaceutical production from roots. High-tech bioreactor-derived bioactive phytomolecules and biopharmaceuticals hold the prospect of providing permanent remedies for improving human well-being.     

Transformed root cultures for biotechnology

This review is concerned with the application of hairy roots, i.e. plant roots formed from plant cells after transformation by Agrobacterium rhizogenes for the production of bioactive compounds. Transformed root cultures have been established from numerous species of dicotyledonous plants. The plants, as well as the main products accumulated in hairy root cultures derived from these plants, are listed in this paper. Data are presented on novel compounds, hitherto detected only in transformed roots but not occurring in the corresponding intact plants. The possible use of hairy root cultures for the over-production of secondary metabolites and biotransformation of chemicals is discussed. In order to enhance the productivity of hairy root cultures, various methods have been derived, and optimized procedures are proposed. They include selection of high-producing clones, elicitation, composition of growth media, culture conditions and genetic approach. Hairy roots usually store secondary metabolites in vacuoles inside the cells. Therefore, several methods have been used to increase the amount of products released into the medium. Unfortunately, no general procedure is known that works in all cases, and the excretion behaviour of hairy root cultures varies from one species to another and even within one species from one clone to another. Special attention is given to the cultivation methods and bioreactor systems for hairy root cultures. Hairy roots are cultivated usually in shake flasks; however, shake flask culture is not suitable for the complex optimization and continuous control of the culture conditions. In this paper, we are going to present bioreactors proposed for the cultivation of hairy roots under more or less controlled conditions. Modifications of typical bacterial bioreactors, i.e. stirred tanks, airlift loop reactors and other constructions, are presented. A very special type of bioreactor providing good conditions for loose root mass multiplication without oxygen or substrate limitations, is the mist bioreactor. Nowadays, it is practically impossible to select the one best bioreactor type for hairy root culture.     

<Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>: recent developments and promising applications

Agrobacterium rhizogenes is the etiological agent for hairy-root disease (also known as root-mat disease). This bacterium induces the neoplastic growth of plant cells that differentiate to form “hairy roots.” Morphologically, A. rhizogenes-induced hairy roots are very similar in structure to wild-type roots with a few notable exceptions: Root hairs are longer, more numerous, and root systems are more branched and exhibit an agravitropic phenotype. Hairy roots are induced by the incorporation of a bacterial-derived segment of DNA transferred (T-DNA) into the chromosome of the plant cell. The expression of genes encoded within the T-DNA promotes the development and production of roots at the site of infection on most dicotyledonous plants. A key characteristic of hairy roots is their ability to grow quickly in the absence of exogenous plant growth regulators. As a result, hairy roots are widely used as a transgenic tool for the production of metabolites and for the study of gene function in plants. Researchers have utilized this tool to study root development and root–biotic interactions, to overexpress proteins and secondary metabolites, to detoxify environmental pollutants, and to increase drought tolerance. In this review, we provide an up-to-date overview of the current knowledge of how A. rhizogenes induces root formation, on the new uses for A. rhizogenes in tissue culture and composite plant production (wild-type shoots with transgenic roots), and the recent development of a disarmed version of A. rhizogenes for stable transgenic plant production.     

Morphological and biochemical characteristics of genetically transformed roots of Scutellaria andrachnoides

Genetically transformed roots (hairy roots) and callus tissue of skullcap (Scutellaria andrachnoides Vved.) were for the first time introduced in the in vitro culture. S. andrachnoides is the endemic plant of the Kyrgyzstan. These cultures were characterized by active and stable growth in the hormone-free liquid Gamborg nutrient medium. The growth rate of undifferentiated callus tissue was higher than that of hairy roots, which were the source of this callus. The composition of secondary metabolites in hairy roots, callus tissue, and also the roots of seedlings and adult S. andrachnoides plants was analyzed. It was found that S. andrachnoides hairy roots and callus culture retained the ability for the synthesis of flavones typical for the roots of intact plants. Substantial quantitative differences in secondary metabolites were observed between the roots of juvenile and adult plants. In the seedling roots, which like hairy roots have no secondary thickening, wogonoside, a wogonin glucuronide, predominated among flavones. In the roots of adult plants growing due to the secondary thickening, balcalin, a baicalein glucuronide, was a dominating flavon. It is proposed to use the large-scale in vitro cultivation of roots and especially the rapidly growing callus tissue of S. andrachnoides with a profitable content of only one group of flavones for the development of the biotechnological method for producing wogonin and creating on its basis a new drug — a valuable anticancer agent of plant origin with selective cytotoxic activity.     

Application of plant tissue cultures in phytoremediation research: Incentives and limitations

The aim of this review is to critically assess the benefits and limitations associated with the use of in vitro plant cell and organ cultures as research tools in phytoremediation studies. Plant tissue cultures such as callus, cell suspensions, and hairy roots are applied frequently in phytoremediation research as model plant systems. In vitro cultures offer a range of experimental advantages in studies aimed at examining the intrinsic metabolic capabilities of plant cells and their capacity for toxicity tolerance. The ability to identify the contributions of plant cells to pollutant uptake and detoxification without interference from microorganisms is of particular significance in the search for fundamental knowledge about plants. However, if the ultimate goal of plant tissue culture experiments is the development of practical phytoremediation technology, the limitations inherent in the use of in vitro cultures as a representative of whole plants in the field must be recognized. The bioavailability of contaminants and the processes of pollutant uptake and metabolite distribution are likely to be substantially different in the two systems; this can lead to qualitative as well as quantitative differences in metabolic profiles and tolerance characteristics. Yet, many studies have demonstrated that plant tissue cultures are an extremely valuable tool in phytoremediation research. The results derived from tissue cultures can be used to predict the responses of plants to environmental contaminants, and to improve the design and thus reduce the cost of subsequent conventional whole plant experiments. Biotechnol. Bioeng. 2009;103: 60–76.     

Mercury detoxification with transgenic plants and other biotechnological breakthroughs for phytoremediation

Summary Phytoremediation, or the use of plants for removal and detoxification of environmental pollutants, has garnered great attention in recent years. This heightened interest is both scientifically, due the fascinating processes utilized by plants for tolerance and removal of harmful compounds, and commercially, as plants represent a more environmentally compatible and less expensive method of site remediation compared to standard approaches. The majority of phytoremediation studies have been with naturally occurring plant species after empirical discovery of their exceptional abilities for such applications. This has led to a growing body of literature and wider acceptance for plants in many aspects of environmental rehabilitation. However, this has occurred with little understanding of their basic biological mechanisms of action or investigation of alternative strategies for enhancing the capabilities of these extraordinary plants. Better understanding of plant physiology, biochemistry and molecular biology in response to specific contaminants is critical for optimization and advancement of phytoremediation. By applying the tools of biotechnology, the potential for plants as an aggressive method of environmental decontamination may be realized. This paper will serve as an introduction to the first Symposium assembled exclusively to review the use of molecular genetic and biotechnological methods for improvement of plants for phytoremediation. After a brief review of the other invited speakers' works (with more extensive papers following), the pioneering work using bacterial genes expressed in plants for removal of mercurial compounds will be surveyed.     

根系分泌物及其在植物修复中的作用

 近年来环境污染日益严重,污染物在土壤植物中的行为引起了人们的高度关注。利用植物去除土壤水体等介质中污染物的植物修复是近10年来兴起的一项安全、廉价的技术,已成为污染生态学和环境生态学的研究热点,它通过植物吸收、根滤、稳定、挥发等方式清除环境中的重金属和有机污染物。国内外有关植物修复的研究报道和概述很多, 但对植物根系分泌物在植物修复中所起的作用及其机理少有述评。 本文从根系分泌物对土壤重金属和土壤有机污染物的去除作用出发,对根系分泌物的种类、数量及其在去除环境污染物中的作用机理和功能地位进行了总结,并借助研究事例对影响植物根系分泌的内外因子,如植物种类、营养胁迫、重金属胁迫、根际环境的理化性质、土壤微生物及其它环境因子进行了讨论。概言之,根系分泌物在修复污染土壤中的重金属途径是多种多样的,主要是通过调节根际pH值、与重金属形成螯合物、络合反应、沉淀、提高土壤微生物数量和活性来改变重金属在根际中的存在形态以及提高重金属的生物有效性,从而减轻它对环境的危害。在清除有机污染物时,根系分泌物中的酶可以对有机污染物进行直接降解,根系分泌物影响下的微生物也可以对有机污染物进行间接降解,且被认为是主要的降解途径。根系分泌物在植物修复过程中确实起着某些重要作用,今后应将这方面的研究重点放在某些特异性根系分泌物植物,尤其是某些重金属超富集植物资源的寻找、筛选上,通过室内实验和野外研究确定其根系分泌物对清除重金属和有机污染物的效率,证实超富集植物根系分泌物的特异性与污染物超富集的内在联系,找到污染土壤生态恢复和治理的有效方法并加以推广应用,如针对性地在被污染地大面积种植此类具特异性根分泌物植物,并辅以营林措施如修剪等,加快生物修复进程,提高修复效率。植物根系分泌物在植物修复过程中所具有的重要生态意义和可能应用前景,为污染生态学和化学生态学之间的联合研究开拓了全新的领域,今后将取得新的突破和重要进展。     

Heavy metal pollution induced due to coal mining effluent on surrounding aquatic ecosystem and its management through naturally occurring aquatic macrophytes

Three aquatic plants Eichhornia crassipes, Lemna minor and Spirodela polyrhhiza were used in laboratory for the removal of heavy metals from the coal mining effluent. Plants were grown singly as well as in combination during 21 days phytoremediation experiment. Results revealed that combination of E. crassipes and L. minor was the most efficient for the removal of heavy metals while E. crassipes was the most efficient in monoculture. Significant correlations between metal concentration in final water and macrophytes were obtained. Translocation factor i.e. ratio of shoot to root metal concentration revealed that metals were largely retained in the roots of aquatic macrophytes. Analytical results showed that plant roots have accumulated heavy metals approximately 10 times of its initial concentration. These plants were also subjected to toxicity assessment and no symptom of metal toxicity was found therefore, this method can be applied on the large scale treatment of waste water where volumes generated are very high and concentrations of pollutants are low.     

The Fatty Acid Profile Analysis of Cyperus laxus Used for Phytoremediation of Soils from Aged Oil Spill-Impacted Sites Revealed That This Is a C18:3 Plant Species

The effect of recalcitrant hydrocarbons on the fatty acid profile from leaf, basal corm, and roots of Cyperus laxus plants cultivated in greenhouse phytoremediation systems of soils from aged oil spill-impacted sites containing from 16 to 340 g/Kg total hydrocarbons (THC) was assessed to investigate if this is a C18:3 species and if the hydrocarbon removal during the phytoremediation process has a relationship with the fatty acid profile of this plant. The fatty acid profile was specific to each vegetative organ and was strongly affected by the hydrocarbons level in the impacted sites. Leaf extracts of plants from uncontaminated soil produced palmitic acid (C16), octadecanoic acid (C18:0), unsaturated oleic acids (C18:1-C18:3), and unsaturated eichosanoic (C20:2-C20:3) acids with a noticeable absence of the unsaturated hexadecatrienoic acid (C16:3); this finding demonstrates, for the first time, that C. laxus is a C18:3 plant. In plants from the phytoremediation systems, the total fatty acid contents in the leaf and the corm were negatively affected by the hydrocarbons presence; however, the effect was positive in root. Interestingly, under contaminated conditions, unusual fatty acids such as odd numbered carbons (C15, C17, C21, and C23) and uncommon unsaturated chains (C20:3n6 and C20:4) were produced together with a remarkable quantity of C22:2 and C24:0 chains in the corm and the leaf. These results demonstrate that weathered hydrocarbons may drastically affect the lipidic composition of C. laxus at the fatty acid level, suggesting that this species adjusts the cover lipid composition in its vegetative organs, mainly in roots, in response to the weathered hydrocarbon presence and uptake during the phytoremediation process.     

Phytoremediation of organochlorine pesticides: Concept,method, and recent developments

Rapid increase in industrialization of world economy in the past century has resulted in significantly high emission of anthropogenic chemicals in the ecosystem. The organochlorine pesticides (OCPs) are a great risk to the global environment and endanger the human health due to their affinity for dispersion, transportation over long distances, and bioaccumulation in the food chain. Phytoremediation is a promising technology that aims to make use of plants and associated bacteria for the treatment of groundwater and soil polluted by these contaminants. Processes known to be involved in phytoremediation of OCPs include phytoaccumulation, rhizoremediation, and phytotransformation. Vegetation has been accounted to considerably amplify OCP elimination from soil, in contrast to non-planted soil, attributable to both, uptake within plant tissues and high microbial degradation of OCP within the root zone. Developing transgenic plants is a promising approach to enhance phytoremediation capabilities. Recent advances in the application of phytoremediation technique for OCPs, including uptake by plants and plant–microbe association in the rhizosphere for the enhanced degradation and mineralization of these pollutants, is presented in this review. Additionally, some attempts to improve this technique using transgenesis and role of certain enzymes are also discussed.     

Overexpression of <Emphasis Type="Italic">AtGRDP2</Emphasis> gene in common bean hairy roots generates vigorous plants with enhanced salt tolerance

Proteins with glycine-rich repeats have been identified in plants, mammalians, fungi, and bacteria. Plant glycine-rich proteins have been associated to stress response. Previously, we reported that the Arabidopsis thaliana AtGRDP2 gene, which encodes a protein with a glycine-rich domain, plays a role in growth and development of A. thaliana and Lactuca sativa. In this study, we generated composite Phaseolus vulgaris plants that overexpress the AtGRDP2 gene in hairy roots generated by Agrobacterium rhizogenes. We observed that hairy roots harboring the AtGRDP2 gene developed more abundant and faster-growing roots than control hairy roots generated with the wild type A. rhizogenes. In addition, composite common bean plants overexpressing the AtGRDP2 gene in roots were more tolerant to salt stress showing increments in their fresh and dry weight. Our data further support the role of plant GRDP genes in development and stress response.     

露水草毛状根的诱导和培养

发根农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍｒｈｉｚｏｇ６ｎｅｓ）ｐＲｉ１５８３４菌株感染露水草（Ｃｙａｎｏｔｉｓａｒａｃｈｎｏｉｄｅａ）的茎和根的外植体及小苗,均诱导出毛状根。毛状很能在不含激素的ＭＳ培养基上生长。用纸电泳和高效薄层层析法,在毛状根中检测到甘露碱。用Ｓｏｕｔｈｅｒｎ印迹杂交,在植物ＤＮＡ中检测到ｒｏｌｅ基因。表明Ｒｉ质粒的Ｔ—ＤＮＡ部分已转移到毛状根细胞的ＤＮＡ中。该毛状根能产生β－蜕皮激素。露水草毛状根的诱导培养在单子叶植物中是一个成功的先例。     

Endophyte-assisted phytoremediation: mechanisms and current application strategies for soil mixed pollutants

Abstract

Phytoremediation uses plants and associated microbes to remove pollutants from the environment and is considered a promising bioremediation method. Compared with well-described single contaminant treatments, the number of studies reporting phytoremediation of soil mixed pollutants has increased recently. Endophytes, including bacteria and fungi, exhibit beneficial traits for the promotion of plant growth, stress alleviation, and biodegradation. Moreover, endophytes either directly or indirectly assist host plants to survive high concentrations of organic and inorganic pollutants in the soil. Endophytic microorganisms can also regulate the plant metabolism in different ways, exhibiting a variety of physiological characteristics. This review summarizes the taxa and physiological properties of endophytic microorganisms that may participate in the detoxification of contaminant mixtures. Furthermore, potential biomolecules that may enhance endophyte mediated phytoremediation are discussed. The practical applications of pollutant-degrading endophytes and current strategies for applying this valuable bio-resource to soil phytoremediation are summarized.     

Dynamics of plant responses to combinations of air pollutants

The focus of this review is on how plants respond to combinations of multiple air pollutants. Global pollution trends, plant physiological responses and ecological perspectives in natural and agricultural systems are all discussed. In particular, we highlight the importance of studying sequential or simultaneous exposure of plants to pollutants, rather than exposure to individual pollutants in isolation, and explore how these responses may interfere with the way plants interact with their biotic community. Air pollutants can alter the normal physiology and metabolic functioning of plants. Here we describe how the phenotypic and molecular changes in response to multiple pollutants can differ compared to those elicited by single pollutants, and how different responses have been observed between plants in the field and in controlled laboratory conditions and between trees and crop plants. From an ecological perspective, we discuss how air pollution can result in greater susceptibility to biotic stressors and in direct or indirect effects on interactions with organisms that occupy higher trophic levels. Finally, we provide an overview of the potential uses of plants to mitigate air pollution, exploring the feasibility for pollution removal via the processes of bio‐accumulation and phytoremediation. We conclude by proposing some new directions for future research in the field.