A quick and efficient system for antibiotic-free expression of heterologous genes in tobacco roots

Requirement for antibiotic-resistance selection markers and difficulty in identifying transgenes with the highest expression levels remain the major obstacles for rapid production of recombinant proteins in plants. An alternative approach to producing transgenic plants free of antibiotic-resistance markers is the phenotypic-based selection with root-proliferation genes (rol genes) of Agrobacterium rhizogenes. By using Agrobacterium tumefaciens harboring the pRYG transformation vector with a cluster of rol genes linked to a heterologous gene of interest, we have developed a rapid transformation tool using hairy root formation as a selection marker. The expression of β-glucuronidase in newly induced transgenic tobacco roots could be detected as early as 12 days after inoculation. Higher levels of transgene expression in the roots correlated positively with the rates of root elongation on hormone-free medium and thus could be used for positive selection. When tobacco plants were transformed with pRYG harboring the expression cassette for secreted alkaline phosphatase (SEAP), the release of SEAP from roots of the fully regenerated transgenic plants could be quantified at rates as high as 28 μg/g root dry weight per day.