Factors affecting Agrobacterium tumefaciens-mediated transformation of peppermint

Substantial improvement in peppermint (Mentha x piperita L. var. Black Mitc ham) genetic transformation has been achieved so that the frequency of tran sgenic plants regenerated (percent of leaf explants that produced transform ed plants) was 20-fold greater than with the original protocol. Essential m odifications were made to conditions for Agrobacterium tumefaciens co-culti vation that enhanced infection, and for selection of transformed cells and propagules during regeneration. A systematic evaluation bf cocultivation pa rameters established that deletion of coconut water from the co-cultivation medium resulted in substantially increased transient beta-Glucuronidase (G US) activity, in both the frequency of explants expressing gusA and the num ber of GUS foci per explant (>700 explants). Co-cultivation on a tobacco ce ll feeder laver also enhanced A. tumefaciens infection. Enhanced transforma tion efficiencies were further facilitated by increased selection pressure mediated by higher concentrations of kanamycin in the medium during shoot i nduction, regeneration, and rooting: from 20 to 50 mg/l in shoot induction/ regeneration medium and from 15 to 30 mg/l in rooting medium. Raising the c oncentration of kanamycin in media substantially lowered the number of "esc apes" without significant reduction in plant regeneration. These modificati ons to the protocol yielded an average transformation frequency of about 20 % (>2000 explants) based on expression of GUS activity or the tobacco antif ungal protein, osmotin, in transgenic plants. Genetic transformation of pep permint has been enhanced to the extent that biotechnology is a viable alte rnative to plant breeding and clonal selection for improvement of this crop .