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. Documents Trust with USADPLC. Log 41182.
3. Progress Report:
This research relates to objective 1 of the associated in-house project “Develop and release new varieties and germplasm of peas, lentils, and chickpea that have higher seed mineral concentrations; improved host-plant resistance to Aphanomyces root rot, Sclerotinia wilt and Ascochyta blight; and higher yields than existing commercial varieties”. New plant expression vectors have been developed that contain several differenct selctable markers. One construct contains a gene that confers tolerance to chlorsulfuron herbicide and another gene that confers tolerance to the antibiotic kanamycin. Several strains of Agrobacterium have been transformed with this construct and growth curves have been determned for all bacterial strains. Use of this construct in plant transformation studies will allow ofor simultaneous selection of both traits during tissue culture. Other plant expression vectors developed include one that has a gene conferring resistance to chlorsulfuron and another gene that allows for GUS expression in transformed tissues.