Direct Agrobacterium tumefaciens-mediated transformation of Hyoscyamus muticus hairy roots using green fluorescent protein

Hyoscyamus muticus hairy root segments were infected with Agrobacterium tum efaciens ASE containing the binary vector pCGN1548 with a green fluorescent protein (GFP) reporter gene under the control of the CaMV 35S promoter. Th e roots were incubated on callus-inducing medium to generate transformed ce lls. Transformants were selected on medium containing 50 and 100 mg/L kanam ycin and screened by visual inspection for GFP expression. Highly fluoresce nt cells were incubated on phytohormone-free medium for regeneration of the hairy root phenotype. This infection technique can be applied directly to existing hairy root cultures which have been previously characterized and s elected for desirable physiological traits. These studies also indicate tha t GFP is not toxic to H. muticus plant tissue and that H. muticus hairy roo ts have minimal autofluorescence which allows for clear observation of GFP.