University of Kentucky
A New Non-Antibiotic Selection System for Plants
Institution
University of Kentucky
Faculty Advisor/ Mentor
David F. Hildebrand
Abstract
There is a need for additional non-antibiotic selection systems for plant genetic engineering. Many effective herbicides are amino acid biosynthetic inhibitors. Studies with lysine and threonine synthesis inhibitors suggest that they might be useful in this regard. Normal plant aspartate kinase (AK) is feed-back inhibited by moderate levels of lysine + threonine, starving the cells of methionine. Plant dihydrodipicolinate synthase (DHPS) is inhibited by the lysine analog S-(2-aminoethyl)-L-cysteine (AEC) killing cells and tissues by blocking lysine synthesis. Natural forms of AK and DHPS are known in bacteria that are resistant to levels of AEC and lysine + threonine that inhibit the corresponding plant enzymes. In order to assess whether AEC and lysine + threonine selection of plants is possible, leaf dip transformation using Agrobacterium tumefaciens was performed. The transformation was confirmed using PCR analysis and staining for a marker gene introduced together with the AK or DHPS. It was found that transgenic samples containing the introduced DHPS were resistant to AEC and samples containing the introduced aspartate kinase were resistant to lysine + threonine. This research not only provides alternative non-antibiotic selection systems for plants but selective agents that are all natural molecules rather than synthetic chemicals.
A New Non-Antibiotic Selection System for Plants
There is a need for additional non-antibiotic selection systems for plant genetic engineering. Many effective herbicides are amino acid biosynthetic inhibitors. Studies with lysine and threonine synthesis inhibitors suggest that they might be useful in this regard. Normal plant aspartate kinase (AK) is feed-back inhibited by moderate levels of lysine + threonine, starving the cells of methionine. Plant dihydrodipicolinate synthase (DHPS) is inhibited by the lysine analog S-(2-aminoethyl)-L-cysteine (AEC) killing cells and tissues by blocking lysine synthesis. Natural forms of AK and DHPS are known in bacteria that are resistant to levels of AEC and lysine + threonine that inhibit the corresponding plant enzymes. In order to assess whether AEC and lysine + threonine selection of plants is possible, leaf dip transformation using Agrobacterium tumefaciens was performed. The transformation was confirmed using PCR analysis and staining for a marker gene introduced together with the AK or DHPS. It was found that transgenic samples containing the introduced DHPS were resistant to AEC and samples containing the introduced aspartate kinase were resistant to lysine + threonine. This research not only provides alternative non-antibiotic selection systems for plants but selective agents that are all natural molecules rather than synthetic chemicals.