Isolation and characterization of cadmium- and lead-accumulating mutants of Brassica juncea (Indian mustard)

Phytoremediation, or the use of plants to remediate metal pollutants, is a cost effective and socially well-received remediation technology. Phytoremediation has been divided into three subsets: phytoextraction (plant-based soil remediation), rhizofiltration (plant-based water remediation) and blastofiltration (seedling-based water remediation). The success of phytoremediation is based on the ability of Brassica juncea (Indian mustard) plants to absorb, concentrate and precipitate toxic metals from soil and water. Plants with genetically enhanced metal uptake would be useful as superior cultivars for this application and would serve as a system for study of the biological mechanisms of metal uptake. The goal of this project was to develop a method for isolating these plants and to study their unique properties.; A mutagenesis-based screening program was developed based on incubating young B. juncea seedlings in solutions containing radioisotopes of Cd and Pb and visualizing their accumulation with a phosphorimager. This high throughput method allowed (1) accurate quantification of metal accumulation; (2) determination of the spatial and temporal distribution of metal in plant parts; and (3) screening for the accumulation of multiple metals simultaneously.; Approximately EMS-mutagenized 50,000 M2 seedlings were screened and 21 putative mutants were recovered with 13 accumulating only Pb, 1 accumulating only Cd and 7 accumulating both Cd and Pb. Many of these lines had no segregation in the M3 generation. All lines were selfed and raised to the M4 generation, seed was collected and growth and accumulation characteristics recorded. Plants of several lines attained a size several times greater than the wild type, which may prove useful for phytoextraction applications.; Pb accumulating mutant 7/15-1 was chosen for further study. Its rate of root Pb accumulation per gram fresh weight was 3.6 times higher than wild type in M4 generation seedlings as determined by phosphorimaging and scintillation counting. Total Pb per root was comparable to wild type. Roots of 7/15-1 were significantly shorter and thicker than wild type roots, with 100% of the M3 and M4 progeny exhibiting this phenotype. Data suggest that this phenotype was not attributed to ethylene perception, but may involve a lesion downstream of perception. Light microscopy of mutant and wild type roots showed that mutant roots were composed of cells which failed to elongate. These smaller cells contained more cell wall material on a per gram fresh weight basis than root cells of wild type, which partially explains their ability to accumulate more Pb. There was a strong correlation between increased root Pb accumulation and decreased root fresh weight and cell size for both mutant and stunted wild type roots, suggesting that root cell architecture is a factor in Pb accumulation.