(a) Field of the Invention
The present invention relates to a method of producing transformants with enhanced heavy metal resistance. More particularly, the present invention relates to transgenic plants that have an improved growth but decreased heavy metal contents when grown in environment contaminated with heavy metals, thus this method can be used for developing plants for phytoremediation and also for developing safe crops.
(b) Description of the Related Art
Heavy metals are major environmental toxicants, which cause reactive oxidation species generation, DNA damage, and enzyme inactivation by binding to active sites of enzymes in cells.
Contamination of the environment with heavy metals has increased drastically due to industrialization. By the early 1990s, the worldwide annual release had reached 22,000 tons of cadmium, 954,000 tons of copper, 796,000 tons of lead, and 1,372,000 tons of zinc (Alloway B J & Ayres D C (1993) Principles of environmental pollution. Chapman and Hall, London). The soils contaminated with heavy metal inhibit normal plant growth and cause contamination of foodstuffs. Many heavy metals are very toxic to human health and carcinogenic at low concentrations. Therefore removal of heavy metals from the environment is an urgent issue.
Studies for removing heavy metals from soil are very actively progressing worldwide. Traditional methods of dealing with soil contaminants include physical and chemical approaches, such as the removal and burial of the contaminated soil, isolation of the contaminated area, fixation (chemical processing of the soil to immobilize the metals), and leaching using an acid or alkali solution (Salt D E, Blaylock M, Kumar N P B A, Viatcheslav D, Ensley B D, et al. (1995). Phytoremediation: a novel strategy for the removal of toxic metals from the environment using plants. Bio-Technology 13,468–74; Raskin I, Smith R D, Salt D E. (1997) Phytoremediation of metals: using plants to remove pollutants from the environment. Curr. Opin. Biotechnol. 8, 221–6). These methods, however, are costly and energy-intensive processes.
Phytoremediation has recently been proposed as a low-cost, environment-friendly way to remove heavy metals from contaminated soils, and is a relatively new technology for cleanup of contaminated soil that uses general plants, specially bred plants, or transgenic plants to accumulate, remove, or detoxify environmental contaminants. The phytoremediation technology is divided into phytoextraction, rhizofiltration, and phytostabilization.
Phytoextraction is a method using metal-accumulating plants to extract metals from soil into the harvestable parts of the plants; rhizofiltration is a method using plant roots to remove contaminants from polluted aqueous streams; and phytostabilization is the stabilization of contaminants such as toxic metals in soils to prevent their entry into ground water, also with plants (Salt et al., Biotechnology 13(5): 468–474, 1995).
Examples of phytoremediation are methods using the plants of Larrea tridentate species that are particularly directed at the decontamination of soils containing copper, nickel, and cadmium (U.S. Pat. No. 5,927,005), and a method using Brassicaceae family (Baker et al., New Phytol. 127:61–68, 1994).
In addition, phytoremediation using transgenic plants that are generated by introducing genes having resistant activity for heavy metals have been attempted. Examples of heavy metal resistant genes are CAX2 (Calcium exchanger 2), cytochrome P450 2E1, NtCBP4 (Nicotiana tabacum calmodulin-binding protein), GSHII (glutathione synthetase), merB (organomercurial lyase), and MRT polypeptide (metal-regulated transporter polypeptide).
CAX2 (Calcium exchanger 2), isolated from Arabidopsis thaliana, accumulates heavy metals including cadmium and manganese in plants (Hirschi et al., Plant Physiol. 124:125–134, 2000). Cytochrome P450 2E1 uptakes and decomposes organic compounds such as trichloroethylene (Doty SL et al., Proc. Natl. Acad. Sci. USA 97:6287–6291, 2000). Nicotiana tabacum transformed with NtCBP4 has resistant activity for nickel (Arazi et al., Plant J. 20:171–182, 1999), GSHII accumulates cadmium (Liang et al., Plant Physiol. 119:73–80,1999), merB detoxifies organic mercury (Bizily et al., Proc. Natl. Acad. Sci. USA 96:6808–6813, 1999), and MRT polypeptide removes heavy metals including cadmium, zinc, and manganese from contaminated soil (U.S. Pat. No. 5,846,821).
However, the transgenic plants generated by introducing the above-mentioned genes have limitations in growth due to accumulation of heavy metals, and they can produce contaminated fruits and crops, when grown in contaminated soil. Therefore, there is a need for plants that have a lower uptake of heavy metals than the wild type, and that maintain healthy growth even in an environment contaminated with heavy metals.