Expression of Genes Conferring Herbicide Resistance in Transgenic Lentil
Grain Legume Genetics Physiology Research
2012 Annual Report
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.
Several different strains of Agrobacterium tumefaciens have been transformed with two different expression vectors, one containing a gene for resistance to glufosinate and the other a gene for resistance to chlorsulfuron. Baseline levels of susceptibility to both herbicides have been determined for several lentil breeding lines in order to know how much of each compound must be added to tissue culture media to allow for the selection of transformed plants. The results of this project relate to Subobjective 1A (Develop pea, lentil and chickpea cultivars with broad adaptation to diverse production environments and with resistance to fungal and viral pathogens) of the in-house associated project.
ARS PI monitoring activities to evaluate research progress included: phone
calls/conference calls, on-site Cooperator/ARS meetings, email communications, discussions at professional conferences/meetings, discussions at annual workshops/conferences, review of Accomplishment Report.