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

Abstract

Hyoscyamus muticus hairy root segments were infected with Agrobacterium tumefaciens ASE containing the binary vector pCGN1548 with a green fluorescent protein (GFP) reporter gene under the control of the CaMV 35S promoter. The roots were incubated on callus-inducing medium to generate transformed cells. Transformants were selected on medium containing 50 and 100 mg/L kanamycin and screened by visual inspection for GFP expression. Highly fluorescent 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 selected for desirable physiological traits. These studies also indicate that GFP is not toxic to H. muticus plant tissue and that H. muticus hairy roots have minimal autofluorescence which allows for clear observation of GFP.