ABSTRACT
Eastern cottonwood (Populus deltoides Bartr. ex Marsh.) trees were engineered to express merA (mercuric ion reductase) and merB (organomercury lyase) transgenes in order to be used for the phytoremediation of mercury-contaminated soils. Earlier studies with Arabidopsis thaliana and Nicotiana tabacum showed that this gene combination resulted in more efficient detoxification of organomercurial compounds than did merB alone, but neither species is optimal for long-term field applications. Leaf discs from in vitro-grown merA, nptII (neomycin phosphotransferase) transgenic cottonwood plantlets were inoculated with Agrobacterium tumefaciens strain C58 carrying the merB and hygromycin resistance (hptII) genes. Polymerase chain reaction of shoots regenerated from the leaf discs under selection indicated an overall transformation frequency of 20%. Western blotting of leaves showed that MerA and MerB proteins were produced. In vitro-grown merA/merB plants were highly resistant to phenylmercuric acetate, and detoxified organic mercury compounds two to three times more rapidly than did controls, as shown by mercury volatilization assay. This indicates that these cottonwood trees are reasonable candidates for the remediation of organomercury-contaminated sites.
Subject(s)
Lyases/genetics , Oxidoreductases/genetics , Phenylmercuric Acetate/metabolism , Populus/genetics , Biodegradation, Environmental , Blotting, Western , Cinnamates/pharmacology , Drug Resistance , Hygromycin B/analogs & derivatives , Hygromycin B/pharmacology , Organomercury Compounds/metabolism , Phenylmercuric Acetate/pharmacology , Plant Leaves/genetics , Plant Leaves/metabolism , Plant Shoots/genetics , Plant Shoots/growth & development , Plants, Genetically Modified , Polymerase Chain Reaction , Populus/enzymology , Populus/growth & development , Rhizobium/genetics , Transformation, Genetic , TransgenesABSTRACT
Black willow (Salix nigra Marsh.) is the largest and only commercially important willow species in North America. It is a candidate for phytoremediation of polluted soils because it is fast-growing and thrives on floodplains throughout eastern USA. Our objective was to develop a protocol for the in vitro regeneration of black willow plants that could serve as target material for gene transformation. Unexpanded inflorescence explants were excised from dormant buds collected from three source trees and cultured on woody plant medium (WPM) supplemented with one of: (1) 0.1 mg l(-1) thidiazuron (TDZ); (2) 0.5 mg l(-1) 6-benzoaminopurine (BAP); or (3) 1 mg l(-1) BAP. All plant growth regulator (PGR) treatments induced direct adventitious bud formation from the genotypes. The percentage of explants producing buds ranged from 20 to 92%, depending on genotype and treatment. Although most of the TDZ-treated inflorescences produced buds, these buds failed to elongate into shoots. Buds on explants treated with BAP elongated into shoots that were easily rooted in vitro and further established in potting mix in high humidity. The PGR treatments significantly affected shoot regeneration frequency (P < 0.01). The highest shoot regeneration frequency (36%) was achieved with Genotype 3 cultured on 0.5 mg l(-1) BAP. Mean number of shoots per explant varied from one to five. The ability of black willow inflorescences to produce adventitious shoots makes them potential targets for Agrobacterium-mediated transformation with heavy-metal-resistant genes for phytoremediation.
