How to produce and characterize transgenic plants

Abstract

A simple and inexpensive protocol to produce and characterize transgenic plants for undergraduate students has been developed. Leaf tissue explants from greenhouse or in vitro-grown petunia or tobacco plants are incubated on a tissue culture medium to induce cell division and expansion. Selected explants are infected with Agrobacterium vir-helper strain EHA105 containing T-DNA vector pYI-mas. Vector pYI-mas contains three genes for the synthesis of novel carbon and nitrogen compounds called the mannityl opines and the plant selectable marker gene conferring antibiotic resistance to the transformed plant cells. Ninety-seven percent of the explants infected with EHA105 (pYI-mas) gave rise to kanamycin resistant callus, of which 18% differentiated a shoot meristem. Over half (57%) of the infected explants produced one or more shoots under antibiotic selection. In four experimental trials, between 16 and 34% of the antibiotic-resistant shoots differentiated a root in the presence of the antibiotic. Characterization of opines by paper electrophoresis confirmed at least one member of the mannityl opine family is produced in 79% of the antibiotic-resistant plants. Undergraduate and independent study students have successfully carried out this instructional unit.