Western Kentucky University
Genetic Modification of a Wild Shrub, Sesbania Drummondii for Environmental Clean Up
Institution
Western Kentucky University
Faculty Advisor/ Mentor
Nilesh Sharma
Abstract
A leguminous shrub, Sesbania drummondii, naturally distributed in coastal areas of the southeast USA, has the ability to grow in heavy metal contaminated areas removing significant amounts of Pb and Hg from soil. The phytoremediation efficiency of this species can be enhanced if it over expresses a natural chelating agent, citrate. As development of a cell regeneration system is a prerequisite for gene transfer, leaf and stem segments were cultured on different basal media fortified with different concentrations and combinations of auxins and cytokinins. Callus proliferation was achieved on several combinations of NAA/Picolinic acid and BAP/ Zeatin or TDZ with the development of a range of callus morphologies and textures. Leaf explants when cultured in MS suspension medium containing Picolinic acid 0.1+ BAP 0.5+ NaCl 30+ Ascorbic Acid 100 mg/L or NAA 1.0+ BAP 1.5+ Ascorbic Acid 100 mg/L proliferated in embryogenic callus. Successive stages of embryogenesis e.g., globular, heart shaped, torpedo and cotyledonary embryos were observed under a Scanning Electron Microscope. These somatic embryos are being cultivated on a range of maturation media for further development including their germination into plantlets. A model for the transfer and expression of a foreign gene was developed in this study using Agrobacterium tumefaciens (pCAMBIA 1305.1 plasmid). Expression of the GUSPlusTM gene in regenerants and callus cells was confirmed by GUSPlusTM assay. Genetic modification of regenerative somatic embryos may serve as an excellent system for over expression of a citrate synthase gene in this species.
Genetic Modification of a Wild Shrub, Sesbania Drummondii for Environmental Clean Up
A leguminous shrub, Sesbania drummondii, naturally distributed in coastal areas of the southeast USA, has the ability to grow in heavy metal contaminated areas removing significant amounts of Pb and Hg from soil. The phytoremediation efficiency of this species can be enhanced if it over expresses a natural chelating agent, citrate. As development of a cell regeneration system is a prerequisite for gene transfer, leaf and stem segments were cultured on different basal media fortified with different concentrations and combinations of auxins and cytokinins. Callus proliferation was achieved on several combinations of NAA/Picolinic acid and BAP/ Zeatin or TDZ with the development of a range of callus morphologies and textures. Leaf explants when cultured in MS suspension medium containing Picolinic acid 0.1+ BAP 0.5+ NaCl 30+ Ascorbic Acid 100 mg/L or NAA 1.0+ BAP 1.5+ Ascorbic Acid 100 mg/L proliferated in embryogenic callus. Successive stages of embryogenesis e.g., globular, heart shaped, torpedo and cotyledonary embryos were observed under a Scanning Electron Microscope. These somatic embryos are being cultivated on a range of maturation media for further development including their germination into plantlets. A model for the transfer and expression of a foreign gene was developed in this study using Agrobacterium tumefaciens (pCAMBIA 1305.1 plasmid). Expression of the GUSPlusTM gene in regenerants and callus cells was confirmed by GUSPlusTM assay. Genetic modification of regenerative somatic embryos may serve as an excellent system for over expression of a citrate synthase gene in this species.