Agrobacterium-mediated genetic transformation of switchgrass

Abstract

Although Agrobacterium tumefaciens has been successfully used to transfer genes to a wide range of plant species, it has received little attention for transformation of forage grasses. Therefore, the objective of the present study was to demonstrate Agrobacterium-mediated transformation of switchgrass (Panicum virgatum L.). The A. tumefaciens strain AGL 1 carrying the binary vector pDM805, coding for the phosphinothricin acetyltransferase (bar) and β-glucuronidase (gus) genes, was utilized in these experiments. Somatic embryos, embryogenic calluses, mature caryopses, and plantlet segments served as target tissues for infection. Treated cultures were selected in the presence of 10 mg L-1 bialaphos and the resultant plantlets were treated with the herbicide Basta [monoammonium 2-amino-4(hydroxymethylphosphinyl)butanoate]. T-DNA delivery efficiency was affected by genotype, explant used and the presence or absence of acetosyringone (3′,5′-dimethoxy-4′-hydroxyacetophenone) during inoculation and cocultivation. Approximately 600 transgenic plants were produced, and transformation efficiencies ranged from 0 to nearly 100%. Stable integration, expression, and inheritance of both transgenes were confirmed by molecular and genetic analyses. Approximately 90% of the tested plants appeared to have only one or two copies of the T-DNA inserts. Controlled crosses between T0 and nontransgenic 'Alamo' plants indicated the expected ratio of 1:1 (transgenic:nontransgenicn) in T1 plants for both transgenes according to a χ2 test at P = 0.05. These results indicate that the Agrobacterium method is effective for transferring foreign genes into switchgrass.