Genetic Modification of Wetland Grasses for Phytoremediation Miha´ly Czako´ a, Xianzhong Fengb, Yuke Heb, Dali Lianga, and La´szlo´ Ma´rtona,* a Department of Biological Sciences, University of South Carolina, 700 Sumter St, Columbia, SC 29208, USA. Fax: 803-777-4002. E-mail: [email protected] b National Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, People’s Republic of China * Author for correspondence and reprint requests Z. Naturforsch. 60c, 285Ð291 (2005) Wetland grasses and grass-like monocots are very important natural remediators of pollu- tants. Their genetic improvement is an important task because introduction of key transgenes can dramatically improve their remediation potential. Tissue culture is prerequisite for ge- netic manipulation, and methods are reported here for in vitro culture and micropropagation of a number of wetland plants of various ecological requirements such as salt marsh, brackish water, riverbanks, and various zones of lakes and ponds, and bogs. The monocots represent numerous genera in various families such as Poaceae, Cyperaceae, Juncaceae, and Typhaceae. The reported species are in various stages of micropropagation and Arundo donax is scaled for mass propagation for selecting elite lines for pytoremediation. Transfer of key genes for mercury phytoremediation into the salt marsh cordgrass (Spartina alterniflora) is also reported here. All but one transgenic lines contained both the organomer- curial lyase (merB) and mercuric reductase (merA) sequences showing that co-introduction into Spartina of two genes from separate Agrobacterium strains is possible. Key words: Cell Culture, Mercury, Phytoremediation, Spartina alterniflora Introduction carry out industrial processes such as phytoreme- “Phytoremediation is the use of plants to par- diation, such grass-like plants are important. Ac- tially or substantially remediate pollutants in con- cordingly, it would be useful to be able to provide taminated soil, sludge, sediment ground water, a method by which such plants could be propa- surface water and waste water” according to the gated even in areas in which plants of these genera Environmental Protection Agency of the USA, are sterile and in a manner that would require encompassing a number of methods for the degra- shorter time, less effort and less area than conven- dation (phyto- and rhizodegradation), removal tional methods. It would also be useful if the (phytoextraction, rhizofiltration and phytovolatili- method is independent of seasons and sustainable zation), or immobilization (hydraulic control and at a high rate of propagation. Furthermore, it phytostabilization) of contaminants. would be useful if a method could be provided Phytoremediation of wetlands is a sensitive is- that permitted better genetic manipulation of wet- sue because the ecological balance and species di- land grasses for enhanced remediation ability. versity is supposed to be retained. Remediation Environmentally important plants were neg- cannot be left to one all-purpose wetland species. lected by plant breeders, however, their remedia- Instead, tissue culture and transgenic technology tion potential has also to be explored. Introduc- needs to be developed for several species from tion of key genes to increase the remediation each habitat concerned. Our laboratory has initi- ability is an obvious approach and significant pro- ated a program for developing such technologies gress has been made in recent years in producing for select species from various wetlands, including genetically modified plants from several plant spe- salt marshes, brackish water, riverbanks, and vari- cies. However, a dependable procedure for tissue ous zones of lakes and ponds, and bogs. culture and plant regeneration is a prerequisite for The monocots represent numerous genera in gene transfer. various families such as Poaceae, Cyperaceae, Jun- Plant regeneration from cultured cells of the caceae, and Typhaceae. Whether used as ornamen- great majority of monocot (mostly graminaceous) tals, sources of energy, or as useful vehicles to species that have been reported so far, is achieved 0939Ð5075/2005/0300Ð0285 $ 06.00 ” 2005 Verlag der Zeitschrift für Naturforschung, Tübingen · http://www.znaturforsch.com · D 286 M. Czako´ et al. · Wetland Grasses and Phytoremediation from callus initiated on high concentrations of a genic lines that are resistant to both organomercu- strong auxin, such as 2,4-dichlorophenoxyacetic rials and ionic mercury salts. acid (2,4-D) (Conger, 1995). Induction of regener- able tissue cultures from monocot species is con- ventionally attempted from immature embryos or Materials and Methods immature inflorescences. Both approaches have Immature inflorescences were collected in Co- been shown to work on Typha glauca, T. angusti- lumbia, Charleston, and Hartsville, South Caro- folia and T. latifolia (Rogers et al., 1998). No re- lina. Explants were disinfected by dilute bleach so- port of tissue culturing of the fourth North Ameri- lution then cultured on primary explant medium can species, T. dominguensis, has been found. DM-8 in the dark at 26Ð28 ∞C. DM-8 medium Tissue culture of only one Juncus species has been (Czako´ and Ma´rton, 2001) contained (in mg lÐ1, reported (Sarma and Rogers, 1998), and no re- unless indicated otherwise): Murashige and Skoog ports of regenerable tissue cultures have been salts [MS (Murashige and Skoog, 1962), Sigma found for species of the grass, Erianthus giganteus, Fine Chemicals] 4,300; Miller’s salt solution [6% sedges of the genera Cyperus and Carex, and bul- (w/v) KH2PO4], 3 ml; myo-inositol, 100; Vitamix rushes of the genus Scirpus and Schoenoplectus. (Ma´rton and Browse, 1991), 2 ml; sucrose, 30,000; Here we report on in vitro culture methods for supplemented with the plant growth regulators forty-eight wetland monocot species. One species adenine hemisulfate, 400 µm; picloram, 0.12; in- in particular, Spartina alterniflora of the salt dole-3-butyric acid, 1; 2,4-dichlorophenoxyacetic marshes of the eastern United States has success- acid, 0.5; isopentenyladenine, 0.5; trans-zeatin, 0.5; fully been transformed with genes of phytoreme- thidiazuron (TDZ) 3.0; and solidified with Phyta- diation importance. Most pollutants are accumu- gel (Sigma Fine Chemicals), 2 g lÐ1. Ð1 lated in wetlands and the plants there act as Medium II1-S contained (in mg l , unless indi- ‘pumps’ for nutrients and metal ions by removing cated otherwise): MS salts, 4,300; (NH4)2SO4, 200; stored elements from the sediment pools. They Miller’s salt solution, 3 ml; myo-inositol, 200; Vita- also can degrade organic pollutants in planta or in mix, 2 ml; l-glutamine, 200; sucrose, 30,000; sup- the rhizosphere by releasing exoenzymes and/or plemented with the plant growth regulator 2,4-di- by enhancing the microbial degradation processes. chlorophenoxyacetic acid, 1; solidified with agar Ð1 Fast and efficient breeding of these plant species (granulated, Fisher Scientific), 7 g l . The pH of requires dependable procedures for tissue culture all tissue culture media was adjusted to 5.8 before sterilization in a pressure cooker (109 ∞C, 35 kPa, and plant regeneration, allowing somaclonal selec- 25 min). tion and transfer of key genes. The secondary medium (0201) contained fewer Plants do not have efficient enzymes for break- plant growth regulators at reduced levels: 0.2 mg ing down organomercurials and for detoxification lÐ1 2,4-D and 0.1 µm TDZ of the plant growth reg- of ionic mercury, therefore introduction of organo- ulators. The tertiary, shoot induction/multiplica- mercurial lyase and mercuric reductase genes into tion medium contained no vitamins or myo-inosi- major wetland plants from a bacterial source ap- tol and only 0.1 µm TDZ as plant growth regulator. pears feasible to increase their mercury remedia- Embryogenic callus culture of S. alterniflora was tion ability. The function of the mercuric reductase maintained on R4 medium (Utomo et al., 2001). (merA) and organomercurial lyase (merB) gene Callus was subcultured every four weeks for main- constructs has been demonstrated in model spe- tenance. Regenerated and rooted plants were sep- cies as well as in rice and trees (Rugh et al., 1995, arated, potted in the greenhouse, and initially kept 1998a, 1998b; Heaton et al., 1998, 2003; Bizily under plastic wrap cover to help acclimatization. et al., 1999, 2000). In the binary vectors pVSTImerApe9 (Rugh Spartina alterniflora is an attractive target spe- et al., 1996) and pVSTImerB (Bizily et al., 1999) cies because it is a globally widespread, large, pe- the coding sequence of merA and merB, respec- rennial grass, which forms monocultural stands in tively, is under the control of the cauliflower mo- salt marshes (Mobberley, 1956). Here we report saic virus 35S RNA promoter (p35S::merApe9::ter on transferring and co-expression of the merA and and p35S::merB::ter, respectively, Fig. 1), and there merB genes in S. alterniflora. Both genes have is also a kanamycin resistance gene under the been transferred simultaneously to create trans- control of the nopaline synthase promoter M. Czako´ et al. · Wetland Grasses and Phytoremediation 287 (pNOS::NptII::ter) in the T-region. Further experi- pared (data not shown). Only the medium desig- mental details have been published elsewhere. nated as II1-S supported callus formation. There was no essential difference between callus origi- Results and Discussion nated from