Characterization of the Metallohistin cDNA AgNt84 and Pteris vittata Tissue Culture for Phytoremediation
Date of Award
Master of Science
C. Neal Stewart
Beth Mullin, Zong-Ming Cheng, Arnold Saxton
Contamination of soils with toxic metals such as arsenic and cadmium has become a major environmental and human health risk. Phytoremediation provides a method to remove contaminants from soils that is not only economically viable but also environmentally sound. Metallohistins are proteins that have the capability to bind divalent metal ions such as Ni2+, Zn2+, Co2+, Cu2+ and Cd2+. In this study, a concatemer sequence was designed to try to increase the presence of metal-binding proteins in transgenic plants. Two methods to increase translational efficiency of the metallohistin protein were used: 1) characterization of the full-length metallohistin AgNt84 gene, and 2) construction of three vectors containing different fragments of the AgNt84 cDNA which were transformed into Nicotiana tabacum. The concatemer sequence proved toxic to Escherichia coli cells and could not be cloned into vectors for plant transformation. Explants genetically transformed with vectors containing either the entire AgNt84 cDNA or the 5’ untranslated and coding region of the cDNA recovered from tissue culture. Explants genetically transformed with a vector containing only the coding region of the cDNA produced shoots but not roots in tissue culture, and then became necrotic. Characterization of the transformants is underway. The first exon and portion of the intron of the gene has been sequenced.
Phytosensors that can recognize and report the presence of arsenic would provide remediators with a management tool for phytoremediation. A transmission and scanning electron microscopy study of Pteris vittata tissue culture revealed callus formation on epidermal cells of gametophytes, presence of an extracellular matrix on calli, and the formation of croziers during differentiation. Calli induced on semi-solid medium consisted of distinct meristematic nodules. These nodules differentiated randomly, and are unfit for genetic transformation. A new differentiation medium is also described.
A preliminary genetic transformation study was successful in creating protoplasts from both Pteris vittata gametophytes and sporophytes, but unsuccessful with biolistic bombardment of calli. Low yields, cellular debris, and autofluorescence exhibited by the protoplasts hampered polyethylene glycol-mediated genetic transformation and detection of transgene expression.
Joyce, Blake Lee, "Characterization of the Metallohistin cDNA AgNt84 and Pteris vittata Tissue Culture for Phytoremediation. " Master's Thesis, University of Tennessee, 2008.