Negative selection driven by cytosine deaminase gene in Lycopersicon esculentum hairy roots

The bacterial cytosine deaminase (cd) gene was employed as a negative selec tion marker in transgenic tomato (Lycopersicon esculentum var. cerasiforme) hairy roots. The ed gene was introduced in tomato cells using a modified b inary vector in the Agrobacterium rhizogenes wild type strain 8196. The tra nsconjugant strain was obtained through triparental mating and then inocula ted on tomato stem fragments. The roots that appeared at the inoculation si te were isolated and subcultured until axenic root clones were established. These roots were tested for their ability to grow in medium containing 5-f luorocytosine (5-FC), which becomes toxic in the presence of cytosine deami nase. The data, together with molecular analysis of the CD-T-DNA transferre d, clearly show that expression of ed in root cells provides a strong negat ive selection, completely inhibiting root growth. This negative selection w as obtained in roots where only one copy of the ed gene was integrated in t he cell genome, providing a potential tool for mutagenesis and transposon m obilization studies. (C) 1999 Elsevier Science Ireland Ltd. All rights rese rved.