Direct creation of marker-free tobacco plants from agroinfiltrated leaf discs

Agroinfiltration is employed as a fast way to directly create marker-free transgenic tobacco plants. As an example for the efficiency of the method, Agrobacterium cells harboring a marker-free vector coding for beta-glucuronidase (GUS) were infiltrated into the leaf discs of Nicotiana tabacum, which were then used as explants for marker-free plant regeneration by tissue culture. Through GUS staining, a large number of small calli were shown to be stably transformed on the treated leaf discs at 17 days after agroinfiltration. Most importantly, after continuous culture of the leaf discs until shoot regeneration, about 15% of the regenerants were proven to be transformants by polymerase chain reaction (PCR) analysis.