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

The bacterial cytosine deaminase (cd) gene was employed as a negative selection marker in transgenic tomato (Lycopersicon esculentum var. cerasiforme) hairy roots. The cd gene was introduced in tomato cells using a modified binary vector in the Agrobacterium rhizogenes wild type strain 8196. The transconjugant strain was obtained through triparental mating and then inoculated on tomato stem fragments. The roots that appeared at the inoculation site were isolated and subcultured until axenic root clones were established. These roots were tested for their ability to grow in medium containing 5-fluorocytosine (5-FC), which becomes toxic in the presence of cytosine deaminase. The data, together with molecular analysis of the CD-T-DNA transferred, clearly show that expression of cd in root cells provides a strong negative selection, completely inhibiting root growth. This negative selection was obtained in roots where only one copy of the cd gene was integrated in the cell genome, providing a potential tool for mutagenesis and transposon mobilization studies.