Stability of a foreign gene in transgenic Nicotiana tabacum L. plants during a cycle of dedifferentiation/differentiation

Protoplasts of Nicotiana tabacum were transformed with the APH(3′)II gene, which confers kanamycin resistance. Plants resistant to kanamycin were differentiated and 3 of them were chosen at random. These were used to study the stability of the foreigngene after acycle of dedifferentiation, to produce calli, and differentiation, to produce new plants. The effect of the selective pressure was analyzed by performing dedifferentiation and differentiation in the presence or absence of kanamycin. Inbred plants were also produced from the original transformed plants and used as control. Southern blot analysis of DNA extracted from 66 regenerated plants showed in all cases that no detectable alteration occurred both in gene structure and insertion site. Furthermore the specific activity of the APH(3′)II enzyme was shown to be at high level in all regenerated plants regardless of the fact that they were regenerated in the presence or absence of kanamycin. The results described here are experimental evidence that a hybrid forcing gene is rather stable in a heterologous genome even after dedifferentiation of the transformed plants and differentiation in vitro, i.e. in those conditions known to be correlated with extensive somaclonal variation.