1a.Objectives (from AD-416)
Develop transgenic lentils that express genes that confer resistance to glufosinate and sulfonylurea herbicides and evaluate transgenic lines for resistance to these herbicides.
1b.Approach (from AD-416)
Lentil seeds will be surface sterilized and plated on water agar to germinate. Cotyledonary explants will be excised from seedlings and transformed using isolates of Agrobacterium tumefaciens that harbor binary plasmids that contain a selectable marker gene and genes that confer resistance to either glufosinate or sulfonylurea herbicides. After the cotyledonary explants have been co-cultivated with A. tumefaciens they will be plated on media containing kanamycin and growth regulators to select for transformed shoots. Shoots will be transferred to media to induce rooting. These putative transgenic events will be subjected to PCR to confirm the incorporation of transgenes into the plant genomes. RNA will be extracted from transgenic plants and subjected to reverse transcriptase real-time PCR to compare expression levels of transgenes among transgenic plants. Transgenic plants will be selfed in the growth chamber to produce T1 families. T1 families will be exposed to the herbicides glufosinate and chlorsulfuron and reaction will be assessed. The relationships between transgene expression and herbicide tolerance will be determined.
The gene for resistance to sulfonylurea herbicides has been cloned into a plant expression vector. Methods have been developed for delivering DNA to lentil explants through biolistics. Initial experiments to transform a Spanish Brown lentil line and a Richlea lentil line with this gene construct are being conducted. The lead scientist monitors the Cooperator’s performance through regular phone conversations and email correspondence, and also through meetings held after the field season is completed.