A negative selection scheme for tobacco protoplast-derived cells expressing the T-DNA gene 2

The amido hydrolase encoded by the T-DNA gene 2 catalyzes the conversion of indole-acetamide, α-naphthalene acetamide, and other substrate analogues into the corresponding auxins. As a result, only gene 2-expressing protoplast-derived tobacco cells can grow in medium containing low concentrations (0.2-1 μM) of α-naphthalene acetamide as auxin precursor. However, in a mixture of SR1 and SR1, gene 2 (+) protoplast-derived cells, cross-feeding occurs and consequently no positive selection for gene 2 is obtained. A 100-times higher concentration of α-naphthalene acetamide (between 30 and 300 μM) provides a negative selection scheme. Only the tobacco cells expressing gene 2 are sensitive to the high naphthalene acetamide concentration and cannot grow to colonies, while cells lacking the gene 2 product regenerate calli even in mixed gene 2 (+) and gene 2 (-) cell populations. Thus, gene 2 might provide a unique biochemically defined marker to investigate mutations and gene inactivation.